Porcine animals lacking any expression of functional alpha 1, 3 galactosyltransferase

ABSTRACT

The present invention is a porcine animal, tissue, organ, cells and cell lines, which lack any expression of functional alpha 1,3 galactosyltransferase (alpha1,3GT). These animals, tissues, organs and cells can be used in xenotransplantation and for other medical purposes.

This application claims priority to U.S. provisional patent applicationNo. 60/404,775 filed Aug. 21, 2002.

FIELD OF THE INVENTION

The present invention are porcine animals, tissue and organs as well ascells and cell lines derived from such animals, tissue and organs, whichlack any expression of functional alpha 1,3 galactosyltransferase(alpha1,3GT). Such animals, tissues, organs and cells can be used inresearch and in medical therapy, including in xenotransplantation.

BACKGROUND OF THE INVENTION

Patients with end stage organ failure require organ transplantation forsurvival. The major limiting factor in clinical transplantation is theshortage of suitable human donors. Over the past ten years the size ofthe waiting list of patients for organs has increased dramatically, fromapproximately 30,000 in 1991 to approximately 80,000 in 2001 (Source:New York Organ Donor Network; Association of Organ ProcurementOrganizations' Death Record Review Study from 1997 to 1999, provided by30 organ procurement organizations). Despite this increasing need overthe past ten years, the number of organ donations has remained flat(approximately 20,000 per year).

According to the United Network for Organ Sharing (UNOS) as of Jul. 17,2003, there were 82,249 patients waiting for organ transplants in theUnited States. The need for specific organs was as follows:

Kidney 55,133 Liver 17,304 Pancreas 1,413 Kidney and 2,378 PancreasIntestine 173 Heart 3,717 Heart-Lung 184 Lung 3,912

Across the U.S., an average of 17 men, women and children of all racesand ethnic backgrounds die every day for lack of donated organs, thus,each year, more than 6,200 Americans die waiting for an organtransplant. A need for a more reliable and unlimited source of organshas led to investigation of the potential for transplantation of organsfrom other animals, referred to as xenotransplantation.

Pigs are considered the most likely source of xenograft organs. Thesupply of pigs is plentiful, breeding programs are well established, andtheir size and physiology are compatible with humans.Xenotransplantation, however, presents its own set of problems. The mostsignificant is immune rejection. The first immunological hurdle is“hyperacute rejection” (HAR). HAR can be defined by the ubiquitouspresence of high titers of pre-formed natural antibodies binding to theforeign tissue. The binding of these natural antibodies to targetepitopes on the donor organ endothelium is believed to be the initiatingevent in HAR. This binding, within minutes of perfusion of the donororgan with the recipient blood, is followed by complement activation,platelet and fibrin deposition, and ultimately by interstitial edema andhemorrhage in the donor organ, all of which cause failure of the organin the recipient (Strahan et al. (1996) Frontiers in Bioscience 1,e34-41).

Except for Old World monkeys, apes and humans, most mammals carryglycoproteins on their cell surfaces that contain galactose alpha1,3-galactose (Galili et al., J. Biol. Chem. 263: 17755-17762, 1988).Humans, apes and Old World monkeys have a naturally occurring anti-alphagal antibody that is produced in high quantity (Cooper et al., Lancet342:682-683, 1993). It binds specifically to glycoproteins andglycolipids bearing galactose alpha-1,3 galactose.

In contrast, glycoproteins that contain galactose alpha 1,3-galactoseare found in large amounts on cells of other mammals, such as pigs. Thisdifferential distribution of the “alpha-1,3 GT epitope” and anti-Galantibodies (i.e., antibodies binding to glycoproteins and glycolipidsbearing galactose alpha-1,3 galactose) in mammals is the result of anevolutionary process which selected for species with inactivated (i.e.mutated) alpha-1,3-galactosyltransferase in ancestral Old World primatesand humans. Thus, humans are “natural knockouts” of alpha1,3GT. A directoutcome of this event is the rejection of xenografts, such as therejection of pig organs transplanted into humans initially via HAR.

A variety of strategies have been implemented to eliminate or modulatethe anti-Gal humoral response caused by porcine xenotransplantation,including enzymatic removal of the epitope with alpha-galactosidases(Stone et al., Transplantation 63: 640-645, 1997), specific anti-galantibody removal (Ye et al., Transplantation 58: 330-337,1994), cappingof the epitope with other carbohydrate moieties, which failed toeliminate alpha-1,3-GT expression (Tanemura et al., J. Biol. Chem.27321: 16421-16425, 1998 and Koike et al., Xenotransplantation 4:147-153, 1997) and the introduction of complement inhibitory proteins(Dalmasso et al., Clin.Exp.Immunol. 86: 31-35, 1991, Dalmasso et al.Transplantation 52:530-533 (1991)). C. Costa et al. (FASEB J 13, 1762(1999)) reported that competitive inhibition of alpha-1,3-GT inH-transferase transgenic pigs results in only partial reduction inepitope numbers. Similarly, S. Miyagawa et al. (J. Biol. Chem 276, 39310(2001)) reported that attempts to block expression of gal epitopes inN-acetylglucosaminyltransferase III transgenic pigs also resulted inonly partial reduction of gal epitopes numbers and failed tosignificantly extend graft survival in primate recipients.

Single allele knockouts of the alpha-1,3-GT locus in porcine cells andlive animals have been reported. Denning et al. (Nature Biotechnology19: 559-562, 2001) reported the targeted gene deletion of one allele ofthe alpha-1,3-GT gene in sheep. Harrison et al. (Transgenics Research11: 143-150, 2002) reported the production of heterozygous alpha-1,3-GTknock out somatic porcine fetal fibroblasts cells. In 2002, Lai et al.(Science 295: 1089-1092, 2002) and Dai et al. (Nature Biotechnology 20:251-255, 2002) reported the production of pigs, in which one allele ofthe alpha-1,3-GT gene was successfully rendered inactive. Ramsoondar etal. (Biol of Reproduc 69, 437-445 (2003) reported the generation ofheterozygous alpha-1,3-GT knockout pigs that also express humanalpha-1,2-fucosyltransferase (HT), which expressed both the HT andalpha-1,3-GT epitopes.

PCT publication No. WO 94/21799 and U.S. Pat. No. 5,821,117 to theAustin Research Institute; PCT publication No. WO 95/20661 to Bresatec;and PCT publication No. WO 95/28412, U.S. Pat. No. 6,153,428, U.S. Pat.No. 6,413,769 and US publication No. 2003/0014770 to BioTransplant, Inc.and The General Hospital Corporation provide a discussion of theproduction of alpha-1,3-GT negative porcine cells based on knowledge ofthe cDNA of the alpha-1,3-GT gene (and without knowledge of the genomicorganization or sequence). However, there was no evidence that suchcells were actually produced prior to the filing date of theseapplications and the Examples were all prophetic.

The first public disclosure of the successful production of aheterozygous alpha-1,3-GT negative porcine cell occurred in July 1999 atthe Lake Tahoe Transgenic Animal Conference (David Ayares, et al., PPLTherapeutics, Inc.). Prior to the present invention, no one hadpublished or publicly disclosed the production of a homozygous alpha1,3GT negative porcine cell. Further, since porcine embryonic stem cellshave not been available to date, there was and still is no way to use analpha-1,3-GT homogygous embryonic stem cell to attempt to prepare a livehomogygous alpha-1,3-GT knock out pig.

On Feb. 27, 2003, Sharma et al. (Transplantation 75:430-436 (2003)published a report demonstrating a successful production of fetal pigfibroblast cells homozygous for the knockout of the alpha-1,3-GT gene.

PCT publication No. WO 00/51424 to PPL Therapeutics describes thegenetic modification of somatic cells for nuclear transfer. This patentapplication discloses the genetic disruption of the alpha-1,3-GT gene inporcine somatic cells, and the subsequent use of the nucleus of thesecells lacking at least one copy of the alpha-1,3-GT gene for nucleartransfer.

U.S. Pat. No. 6,331,658 to Cooper & Koren claims but does not confirmany actual production of genetically engineered mammals that express asialyltransferase or a fucosyltransferase protein. The patent assertsthat the genetically engineered mammals would exhibit a reduction ofgalactosylated protein epitopes on the cell surface of the mammal.

PCT publication No. WO 03/055302 to The Curators of the University ofMissouri confirms the production of heterozygous alpha 1,3GT knockoutminiature swine for use in xenotransplantation. This application isgenerally directed to a knockout swine that includes a disruptedalpha-1,3-GT gene, wherein expression of functional alpha-1,3-GT in theknockout swine is decreased as compared to the wildtype. Thisapplication does not provide any guidance as to what extent thealpha-1,3-GT must be decreased such that the swine is useful forxenotransplantation. Further, this application does not provide anyproof that the heterozygous pigs that were produced exhibited adecreased expression of functional alpha1,3GT. Further, while theapplication refers to homozygous alpha 1,3GT knockout swine, there is noevidence in the application that any were actually produced orproducible, much less whether the resultant offspring would be viable orphenotypically useful for xenotransplantation.

Total depletion of the glycoproteins that contain galactose alpha1,3-galactose is clearly the best approach for the production of porcineanimals for xenotransplantation. It is theoretically possible thatdouble knockouts, or the disruption of both copies of the alpha 1,3GTgene, could be produced by two methods: 1) breeding of two single alleleknockout animals to produce progeny, in which case, one would predictbased on Mendelian genetics that one in four should be double knockoutsor 2) genetic modification of the second allele in a cell with apre-existing single knockout. In fact, this has been quite difficult asillustrated by the fact that while the first patent application onknock-out porcine cells was filed in 1993, the first homozygous alpha1,3GT knock out pig was not produced until July 2002 (which was based onthe work of the present inventor and described herein).

Transgenic mice (not pigs) have historically been the preferred model tostudy the effects of genetic modifications on mammalian physiology, fora number of reasons, not the least of which is that mouse embryonic stemcells have been available while porcine embryonic stem cells have notbeen available. Mice are ideal animals for basic research applicationsbecause they are relatively easy to handle, they reproduce rapidly, andthey can be genetically manipulated at the molecular level. Scientistsuse the mouse models to study the molecular pathologies of a variety ofgenetically based diseases, from colon cancer to mental retardation.Thousands of genetically modified mice have been created to date. A“Mouse Knockout and Mutation Database” has been created by BioMedNet toprovide a comprehensive database of phenotypic and genotypic informationon mouse knockouts and classical mutations Brandon et al Current Biology5[7]:758-765 (1995); Brandon et al Current Biology 5[8]:873-881 (1995),this database provides information on over 3,000 unique genes, whichhave been targeted in the mouse genome to date.

Based on this extensive experience with mice, it has been learned thattransgenic technology has some significant limitations. Because ofdevelopmental defects, many genetically modified mice, especially nullmice created by gene knock out technology die as embryos before theresearcher has a chance to use the model for experimentation. Even ifthe mice survive, they can develop significantly altered phenotypes,which can render them severely disabled, deformed or debilitated (Pray,Leslie, The Scientist 16 [13]: 34 (2002); Smith, The Scientist14[15]:32, (2000); Brandon et al Current Biology 5[6]:625-634 (1995);Brandon et al Current Biology 5[7]:758-765 (1995); Brandon et al CurrentBiology 5[8]:873-881 (1995). Further, it has been learned that it is notpossible to predict whether or not a given gene plays a critical role inthe development of the organism, and, thus, whether elimination of thegene will result in a lethal or altered phenotype, until the knockouthas been successfully created and viable offspring are produced.

Mice have been genetically modified to eliminate functional alpha-1,3-GTexpression. Double-knockout alpha-1,3-GT mice have been produced. Theyare developmentally viable and have normal organs (Thall et al. J BiolChem 270:21437-40(1995); Tearle et al. Transplantation 61:13-19 (1996),see also U.S. Pat. No. 5,849,991). However, two phenotypic abnormalitiesin these mice were apparent. First, all mice develop dense corticalcataracts. Second, the elimination of both alleles of the alpha-1,3-GTgene significantly affected the development of the mice. The mating ofmice heterozygous for the alpha-1,3-GT gene produced genotype ratiosthat deviated significantly from the predicted Mendelian 1:2:1 ratio(Tearle et al. Transplantation 61:13-19 (1996)).

Pigs have a level of cell surface glycoproteins containing galactosealpha 1,3-galactose that is 100-1000 fold higher than found in mice.(Sharma et al. Transplantation 75:430-436 (2003); Galili et al.Transplantation 69:187-190 (2000)). Thus, alpha1,3-GT activity is morecritical and more abundant in the pig than the mouse.

Despite predictions and prophetic statements, prior to this invention,no one knew whether the disruption of both alleles of the alpha-1,3-GTgene would be lethal or would effect porcine development or result in analtered phenotype (Ayares et al. Graft 4(1)80-85 (2001); Sharma et al.Transplantation 75:430-436 (2003); Porter & Dallman Transplantation64:1227-1235 (1997); Galili, U. Biochimie 83:557-563 (2001)). Indeed,many experts in the field expressed serious doubts as to whetherhomozygous alpha-1,3-GT knockout pigs would be viable at all, much lessdevelop normally. Such concerns were expressed up until the doubleknockout pig of the present invention was produced. Examples ofstatements by those working in the field at the time included thefollowing.

“The abundantly expressed alpha-gal epitope may have some biologicalroles in pig development, such as in cell-cell interaction. If thisassumption is correct, pigs may not develop in the absence of thisepitope (Galili, U. Biochimie 83:557-563 (2001).”

“The inability to generate knockout pigs for alpha-gal may suggest thatalpha-gal epitopes are indispensable in this species (Galili et al.Transplantation 69:187-190 (2000)).”

“Although double-knockout alpha-gal mice develop and remain fairlynormal, the possibility exists that deletion of this enzyme could havemore severe consequences in other animals (Porter & DallmanTransplantation 64:1227-1235 (1997)).”

“It is possible that the GT(−/−) pig may not be viable because the GTgene is essential for embryonic development. An answer to this questionand to the relevance of GT(−/−) pigs to xenotransplantation researchmust await, if possible, the production of the appropriate pigs (Sharmaet al. Transplantation 75:430-436 (2003)).”

“Since Gal epitope expression in pig organs is up to 500-fold higherthan in mouse organs, there is the possibility that alphaGT activity ismore crucial to the pig and could effect development of these pigs(Ayares et al. Graft 4(1)80-85 (2001)).”

Thus, until a viable double alpha-1,3-GT knockout pig is produced,according to those of skill in the art at the time, it was not possibleto determine (i) whether the offspring would be viable or (ii) whetherthe offspring would display a phenotype that allows the use of theorgans for transplantation into humans.

It is therefore an object of the present invention to provide viablepigs which lack any expression of functional alpha1,3GT.

It is another object of the present invention to provide procine cells,tissues and organs, which lack any expression of functional alpha1,3GT,for use in xenotransplantation or other biomedical applications.

It is a further object of the present invention to provide a method toselect and screen for porcine cells, which lack galactose alpha1,3-galactose epitopes on the cell surface.

SUMMARY OF THE INVENTION

This invention is the production of the first live pigs lacking anyfunctional expression of alpha 1,3 galactosyltransferase. The subject ofthis invention was heralded in a full paper in Science magazine in 2003(Phelps et al. (Science 299:411-414 (2003)) and widely reported in thepress as a breakthrough in xenotransplantation.

It has for the first time been proven that a viable porcine animal thatlacks any expression of functional alpha 1,3 galactosyltransferase canbe produced. The present invention provides the complete inactivation ofboth alleles of the alpha 1,3 galactosyltransferase gene in pigs, thusovercoming this longstanding hurdle and making xenotransplantation areality. Eliminating the expression of this gene, resulting in a lack ofgalactose alpha 1,3-galactose epitopes on the cell surface, representsthe first and major step in eliminating hyperacute rejection inpig-to-human xenotransplantation therapy. The invention also providesorgans, tissues, and cells derived from such porcine animals, which areuseful for xenotransplantation.

In embodiments of the present invention, the alleles of the alpha-1,3-GTgene are rendered inactive, such that the resultant alpha-1,3-GT enzymecan no longer generate galactose alpha1,3-galactose on the cell surface.In one embodiment, the alpha-1,3-GT gene can be transcribed into RNA,but not translated into protein. In another embodiment, the alpha-1,3-GTgene can be transcribed in an inactive truncated form. Such a truncatedRNA may either not be translated or can be translated into anonfunctional protein. In an alternative embodiment, the alpha-1,3-GTgene can be inactivated in such a way that no transcription of the geneoccurs. In a further embodiment, the alpha-1,3-GT gene can betranscribed and then translated into a nonfunctional protein.

In another embodiment, pigs that lack any expression of functionalalpha-1,3-GT are useful for providing a clearer evaluation of approachescurrently in development aimed at overcoming potential delayed andchronic rejection mechanisms in porcine xenotransplantation.

In one aspect of the present invention, porcine animals are provided inwhich at least one allele of the alpha-1,3-GT gene is inactivated via agenetic targeting event. In another aspect of the present invention,porcine animals are provided in which both alleles of the alpha-1,3-GTgene are inactivated via a genetic targeting event. The gene can betargeted via homologous recombination. In other embodiments, the genecan be disrupted, i.e. a portion of the genetic code can be altered,thereby affecting transcription and/or translation of that segment ofthe gene. For example, disruption of a gene can occur throughsubstitution, deletion (“knockout”) or insertion (“knockin”) techniques.Additional genes for a desired protein or regulatory sequence thatmodulate transcription of an existing sequence can be inserted.

Pigs that possess two inactive alleles of the alpha-1,3-GT gene are notnaturally occurring. The predicted frequency of occurrence of such a pigwould be in the range of 10⁻¹⁰ to 10⁻¹², and has never been identified.

As one aspect of the invention, it was surprisingly discovered thatwhile attempting to knockout the second allele of the alpha-1,3-GT genethrough a genetic targeting event, a point mutation was identified,which rendered the second allele inactive. Pigs carrying point mutationsin the alpha-1,3-GT gene are free of antibiotic-resistance genes andthus have the potential to make a safer product for human use. Thus,another aspect of the invention is a homozygous alpha-1,3-GT knock outthat has no antibiotic resistant or other selectable marker genes. Inone embodiment, this point mutation can occur via a genetic targetingevent. In another embodiment, this point mutation can be naturallyoccurring. In a further embodiment, mutations can be induced in thealpha-1,3-GT gene via a mutagenic agent.

In one specific embodiment the point mutation can be a T-to-G mutationat the second base of exon 9 of the alpha-1,3-GT gene (FIG. 2). In otherembodiments, at least two, at least three, at least four, at least five,at least ten or at least twenty point mutations can exist to render thealpha-1,3-GT gene inactive. In other embodiments, pigs are provided inwhich both alleles of the alpha-1,3-GT gene contain point mutations thatprevent any expression of functional alpha1,3GT. In a specificembodiment, pigs are provided that contain the T-to-G mutation at thesecond base of exon 9 in both alleles of the alpha-1,3-GT gene (FIG. 2).

Another aspect of the present invention provides a porcine animal, inwhich both alleles of the alpha-1,3-GT gene are inactivated, whereby oneallele is inactivated by a genetic targeting event and the other alleleis inactivated via a point mutation. In one embodiment, a porcine animalis provided, in which both alleles of the alpha-1,3-GT gene areinactivated, whereby one allele is inactivated by a genetic targetingevent and the other allele is inactivated due to presence of a T-to-Gpoint mutation at the second base of exon 9. In a specific embodiment, aporcine animal is provided, in which both alleles of the alpha-1,3-GTgene are inactivated, whereby one allele is inactivated via a targetingconstruct directed to Exon 9 (see, for example, FIG. 6) and the otherallele is inactivated due to presence of a T-to-G point mutation at thesecond base of exon 9 (FIG. 2). Targeting, for example, can also bedirected to exon 9, and or exons 4-8.

In a further embodiment, one allele is inactivated by a genetictargeting event and the other allele is inactivated due to presence of aT-to-G point mutation at the second base of exon 9 of the alpha-1,3-GTgene. In a specific embodiment, one allele is inactivated via atargeting construct directed to Exon 9 (see, for example, FIG. 6) andthe other allele is inactivated due to presence of a T-to-G pointmutation at the second base of exon 9 of the alpha-1,3-GT gene. Inanother embodiment, a method to clone such pigs includes: enucleating anoocyte, fusing the oocyte with a donor nucleus from a porcine cell thatlacks expression of functional alpha1,3GT, and implanting the nucleartransfer-derived embryo into a surrogate mother.

In another embodiment, the present invention provides a method forproducing viable pigs that lack any expression of functionalalpha-1,3-GT by breeding a male pig heterozygous for the alpha-1,3-GTgene with a female pig heterozygous for the alpha-1,3-GT gene. In oneembodiment, the pigs are heterozygous due to the genetic modification ofone allele of the alpha-1,3-GT gene to prevent expression of thatallele. In another embodiment, the pigs are heterozygous due to thepresence of a point mutation in one allele of the alpha-1,3-GT gene. Inanother embodiment, the point mutation can be a T-to-G point mutation atthe second base of exon 9 of the alpha-1,3-GT gene. In one specificembodiment, a method to produce a porcine animal that lacks anyexpression of functional alpha-1,3-GT is provided wherein a male pigthat contains a T-to-G point mutation at the second base of exon 9 ofthe alpha-1,3-GT gene is bred with a female pig that contains a T-to-Gpoint mutation at the second base of exon 9 of the alpha-1,3-GT gene, orvise versa.

In another aspect of the present invention, a selection method isprovided for determining whether porcine cells express galactosealpha1,3-galactose on the cell surface. In one embodiment, the selectionprocedure can be based on a bacterial toxin to select for cells thatlack expression of galactose alpha1,3-galactose. In another embodiment,the bacterial toxin, toxin A produced by Clostridium difficile, can beused to select for such cells. Exposure to C. difficile toxin can causerounding of cells that exhibit this epitope on their surface, releasingthe cells from the plate matrix. Both targeted gene knockouts andmutations that disable enzyme function or expression can be detectedusing this selection method. Cells lacking cell surface expression ofthe galactose alpha 1,3-galactose, identified using Toxin A mediatedselection described, or produced using standard methods of geneinactivation including gene targeting, can then be used to produce pigsthat lack expression of functional alpha1,3GT.

Other embodiments of the present invention will be apparent to one ofordinary skill in light of the following description of the invention,the claims and what is known in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the relative lytic effects of complement oncells from fetuses 680B1-4.

FIG. 2 depicts a short segment of the coding region of the alpha-1,3-GTgene (see GenBank Acc. No. L36152) in which the point mutation selectedby Toxin A occurs. Upper sequence occurs in wild type; lower sequenceshows the change due to the point mutation in the second allele.

FIG. 3 is a representation of a 3-dimensional model of the UDP bindingsite of bovine alpha1,3GT. The aromatic ring of the tyrosine residue(foreground, white) can be seen in close proximity to the uracil base ofUDP (grayscale).

FIG. 4 is a photograph of homozygous, alpha-1,3-GT deficient cloned pigsproduced by the methods of the invention, born on Jul. 25, 2002.

FIG. 5 is a graph depicting Anti-alpha-1,3-gal IgM levels before andafter injections of piglet islet-like cell clusters (ICC) inalpha-1,3-GT KO mice. Each mouse received three serial ICC injectionsvia i.p. (200-500 ICC per injection) over 4 days. All three recipientsof wild-type (WT) piglet ICCs showed a significant elevation ofanti-alpha 1,3Gal IgM titer and subsequent return to baseline 4 weeksafter ICC implants. Sera from all three mice injected with alpha-1,3-GTDKO piglet ICCs maintained low baseline values of anti-alpha-1,3-gal IgMtiter during the observation time of 35 days (Phelps et al., Science299: 411-414, 2003, figure S4).

FIG. 6 is a diagram of the porcine alpha-1,3-GT locus, corresponding toalpha-1,3-GT genomic sequences that can be used as 5′ and 3′ arms inalpha-1,3-GT knockout vectors, and the structure of the targeted locusafter homologous recombination. The names of names and positions of theprimers used for 3′PCR and long-range PCR are indicated by short arrows.The short bar indicates the probe used for alpha-1,3-GT Southern blotanalysis. The predicted size of Southern bands with BstEII digestion forboth the endogenous alpha-1,3-GT locus and the alpha-1,3-GT targetedlocus is also indicated.

DETAILED DESCRIPTION OF THE INVENTION

We have now proven that a viable porcine animal that lacks anyexpression of functional alpha 1,3 galactosyltransferase can beproduced. The present invention provides the complete inactivation ofboth alleles of the alpha 1,3 galactosyltransferase gene in pigs, thusovercoming this longstanding hurdle and making xenotransplantation areality. Eliminating the expression of this gene, resulting in a lack ofgalactose alpha 1,3-galactose on the cell surface, represents the firstand major step in eliminating hyperacute rejection in pig-to-humanxenotransplantation therapy. The invention also provides organs,tissues, and cells derived from such porcine, which are useful forxenotransplantation.

In one aspect, the invention provides porcine organs, tissues and/orpurified or substantially pure cells or cell lines obtained from pigsthat lack any expression of functional alpha1,3GT. In anotherembodiment, the invention provides organs or tissues that are useful forxenotransplantation. In a further embodiment, the invention providescells or cell lines that are useful for xenotransplantation.

Definitions

As used herein, the term “animal” (as in “genetically modified (oraltered) animal”) is meant to include any non-human animal, particularlyany non-human mammal, including but not limited to pigs, sheep, goats,cattle (bovine), deer, mules, horses, monkeys, dogs, cats, rats, mice,birds, chickens, reptiles, fish, and insects. In one embodiment of theinvention, genetically altered pigs and methods of production thereofare provided.

As used herein, an “organ” is an organized structure, which can be madeup of one or more tissues. An “organ” performs one or more specificbiological functions. Organs include, without limitation, heart, liver,kidney, pancreas, lung, thyroid, and skin.

As used herein, a “tissue” is an organized structure comprising cellsand the intracellular substances surrounding them. The “tissue”, aloneor in conjunction with other cells or tissues can perform one or morebiological functions.

As used herein, the terms “porcine”, “porcine animal”, “pig” and “swine”are generic terms referring to the same type of animal without regard togender, size, or breed.

I. Genetic Targeting of the Alpha-1,3-GT Gene

In one aspect of the present invention, porcine animals are provided inwhich one allele of the alpha-1,3-GT gene is inactivated via a genetictargeting event. In another aspect of the present invention, porcineanimals are provided in which both alleles of the alpha-1,3-GT gene areinactivated via a genetic targeting event. In one embodiment, the genecan be targeted via homologous recombination. In other embodiments, thegene can be disrupted, i.e. a portion of the genetic code can bealtered, thereby affecting transcription and/or translation of thatsegment of the gene. For example, disruption of a gene can occur throughsubstitution, deletion (“knockout”) or insertion (“knockin”) techniques.Additional genes for a desired protein or regulatory sequence thatmodulate transcription of an existing sequence can be inserted.

In embodiments of the present invention, the alleles of the alpha-1,3-GTgene are rendered inactive, such that the resultant alpha-1,3-GT enzymecan no longer generate galactose alpha1,3-galactose on the cell surface.In one embodiment, the alpha-1,3-GT gene can be transcribed into RNA,but not translated into protein. In another embodiment, the alpha-1,3-GTgene can be transcribed in a trancated form. Such a truncated RNA caneither not be translated or can be translated into a nonfunctionalprotein. In an alternative embodiment, the alpha-1,3-GT gene can beinactivated in such a way that no transcription of the gene occurs. In afurther embodiment, the alpha-1,3-GT gene can be transcribed and thentranslated into a nonfunctional protein.

Pigs that possess two inactive alleles of the alpha-1,3-GT gene are notnaturally occurring. It was surprisingly discovered that whileattempting to knockout the second allele of the alpha-1,3-GT genethrough a genetic targeting event, a point mutation was identified,which prevented the second allele from producing functional alpha1,3GT.

Thus, in another aspect of the present invention, the alpha-1,3-GT genecan be rendered inactive through at least one point mutation. In oneembodiment, one allele of the alpha-1,3-GT gene can be rendered inactivethrough at least one point mutation. In another embodiment, both allelesof the alpha-1,3-GT gene can be rendered inactive through at least onepoint mutation. In one embodiment, this point mutation can occur via agenetic targeting event. In another embodiment, this point mutation canbe naturally occurring. In a further embodiment, mutations can beinduced in the alpha-1,3-GT gene via a mutagenic agent.

In one specific embodiment the point mutation can be a T-to-G mutationat the second base of exon 9 of the alpha-1,3-GT gene (FIG. 2). Pigscarrying a naturally occurring point mutation in the alpha-1,3-GT geneallow for the production of alpha1,3GT-deficient pigs free ofantibiotic-resistance genes and thus have the potential to make a saferproduct for human use. In other embodiments, at least two, at leastthree, at least four, at least five, at least ten or at least twentypoint mutations can exist to render the alpha-1,3-GT gene inactive. Inother embodiments, pigs are provided in which both alleles of thealpha-1,3-GT gene contain point mutations that prevent any expression offunctional alpha1,3GT. In a specific embodiment, pigs are provided thatcontain the T-to-G mutation at the second base of exon 9 in both allelesof the alpha-1,3-GT gene (FIG. 2).

Another aspect of the present invention provides a porcine animal, inwhich both alleles of the alpha-1,3-GT gene are inactivated, whereby oneallele is inactivated by a genetic targeting event and the other alleleis inactivated via a mutation. In one embodiment, a porcine animal isprovided, in which both alleles of the alpha-1,3-GT gene areinactivated, whereby one allele is inactivated by a genetic targetingevent and the other allele is inactivated due to presence of a T-to-Gpoint mutation at the second base of exon 9. In a specific embodiment, aporcine animal is provided, in which both alleles of the alpha-1,3-GTgene are inactivated, whereby one allele is inactivated via a targetingconstruct directed to Exon 9 (see, for example, FIG. 6) and the otherallele is inactivated due to presence of a T-to-G point mutation at thesecond base of exon 9.

Types of Porcine Cells

Porcine cells that can be genetically modified can be obtained from avariety of different organs and tissues such as, but not limited to,skin, mesenchyme, lung, pancreas, heart, intestine, stomach, bladder,blood vessels, kidney, urethra, reproductive organs, and a disaggregatedpreparation of a whole or part of an embryo, fetus, or adult animal. Inone embodiment of the invention, porcine cells can be selected from thegroup consisting of, but not limited to, epithelial cells, fibroblastcells, neural cells, keratinocytes, hematopoietic cells, melanocytes,chondrocytes, lymphocytes (B and T), macrophages, monocytes, mononuclearcells, cardiac muscle cells, other muscle cells, granulosa cells,cumulus cells, epidermal cells, endothelial cells, Islets of Langerhanscells, blood cells, blood precursor cells, bone cells, bone precursorcells, neuronal stem cells, primordial stem cells, hepatocytes,keratinocytes, umbilical vein endothelial cells, aortic endothelialcells, microvascular endothelial cells, fibroblasts, liver stellatecells, aortic smooth muscle cells, cardiac myocytes, neurons, Kupffercells, smooth muscle cells, Schwann cells, and epithelial cells,erythrocytes, platelets, neutrophils, lymphocytes, monocytes,eosinophils, basophils, adipocytes, chondrocytes, pancreatic isletcells, thyroid cells, parathyroid cells, parotid cells, tumor cells,glial cells, astrocytes, red blood cells, white blood cells,macrophages, epithelial cells, somatic cells, pituitary cells, adrenalcells, hair cells, bladder cells, kidney cells, retinal cells, rodcells, cone cells, heart cells, pacemaker cells, spleen cells, antigenpresenting cells, memory cells, T cells, B cells, plasma cells, musclecells, ovarian cells, uterine cells, prostate cells, vaginal epithelialcells, sperm cells, testicular cells, germ cells, egg cells, leydigcells, peritubular cells, sertoli cells, lutein cells, cervical cells,endometrial cells, mammary cells, follicle cells, mucous cells, ciliatedcells, nonkeratinized epithelial cells, keratinized epithelial cells,lung cells, goblet cells, columnar epithelial cells, squamous epithelialcells, osteocytes, osteoblasts, and osteoclasts.

In one alternative embodiment, embryonic stem cells can be used. Anembryonic stem cell line can be employed or embryonic stem cells can beobtained freshly from a host, such as a porcine animal. The cells can begrown on an appropriate fibroblast-feeder layer or grown in the presenceof leukemia inhibiting factor (LIF). In a preferred embodiment, theporcine cells can be fibroblasts; in one specific embodiment, theporcine cells can be fetal fibroblasts. Fibroblast cells are a preferredsomatic cell type because they can be obtained from developing fetusesand adult animals in large quantities. These cells can be easilypropagated in vitro with a rapid doubling time and can be clonallypropagated for use in gene targeting procedures.

Targeting constructs

Homologous Recombination

Homologous recombination permits site-specific modifications inendogenous genes and thus novel alterations can be engineered into thegenome. In homologous recombination, the incoming DNA interacts with andintegrates into a site in the genome that contains a substantiallyhomologous DNA sequence. In non-homologous (“random” or “illicit”)integration, the incoming DNA is not found at a homologous sequence inthe genome but integrates elsewhere, at one of a large number ofpotential locations. In general, studies with higher eukaryotic cellshave revealed that the frequency of homologous recombination is far lessthan the frequency of random integration. The ratio of these frequencieshas direct implications for “gene targeting” which depends onintegration via homologous recombination (i.e. recombination between theexogenous “targeting DNA” and the corresponding “target DNA” in thegenome).

A number of papers describe the use of homologous recombination inmammalian cells. Illustrative of these papers are Kucherlapati et al.,Proc. Natl. Acad. Sci. USA 81:3153-3157, 1984; Kucherlapati et al., Mol.Cell. Bio. 5:714-720, 1985; Smithies et al, Nature 317:230-234, 1985;Wake et al., Mol. Cell. Bio. 8:2080-2089, 1985; Ayares et al., Genetics111:375-388, 1985; Ayares et al., Mol. Cell. Bio. 7:1656-1662, 1986;Song et al., Proc. Natl. Acad. Sci. USA 84:6820-6824, 1987; Thomas etal. Cell 44:419-428, 1986; Thomas and Capecchi, Cell 51: 503-512, 1987;Nandi et al., Proc. Natl. Acad. Sci. USA 85:3845-3849, 1988; and Mansouret al., Nature 336:348-352, 1988. Evans and Kaufman, Nature 294:146-154,1981; Doetschman et al., Nature 330:576-578, 1987; Thoma and Capecchi,Cell 51:503-512,4987; Thompson et al., Cell 56:316-321, 1989.

The present invention uses homologous recombination to inactivate thealpha-1,3-GT gene in cells, such as the porcine cells described above.The DNA can comprise at least a portion of the gene(s) at the particularlocus with introduction of an alteration into at least one, optionallyboth copies, of the native gene(s), so as to prevent expression offunctional alpha1,3GT. The alteration can be an insertion, deletion,replacement or combination thereof. When the alteration is introduceinto only one copy of the gene being inactivated, the cells having asingle unmutated copy of the target gene are amplified and can besubjected to a second targeting step, where the alteration can be thesame or different from the first alteration, usually different, andwhere a deletion, or replacement is involved, can be overlapping atleast a portion of the alteration originally introduced. In this secondtargeting step, a targeting vector with the same arms of homology, butcontaining a different mammalian selectable markers can be used. Theresulting transformants are screened for the absence of a functionaltarget antigen and the DNA of the cell can be further screened to ensurethe absence of a wild-type target gene. Alternatively, homozygosity asto a phenotype can be achieved by breeding hosts heterozygous for themutation.

Targeting Vectors

Modification of a targeted locus of a cell can be produced byintroducing DNA into the cells, where the DNA has homology to the targetlocus and includes a marker gene, allowing for selection of cellscomprising the integrated construct. The homologous DNA in the targetvector will recombine with the chromosomal DNA at the target locus. Themarker gene can be flanked on both sides by homologous DNA sequences, a3′ recombination arm and a 5′ recombination arm. Methods for theconstruction of targeting vectors have been described in the art, see,for example, Dai et al., Nature Biotechnology 20: 251-255, 2002; WO00/51424, FIG. 6.

Various constructs can be prepared for homologous recombination at atarget locus. The construct can include at least 50 bp, 100 bp, 500 bp,1 kbp, 2 kbp, 4 kbp, 5 kbp, 10 kbp, 15 kbp, 20 kbp, or 50 kbp ofsequence homologous with the target locus. The sequence can include anycontiguous sequence of the porcine alpha-1,3-GT gene (see, for example,GenBank Acc. No. L36152, WO0130992 to The University of Pittsburgh ofthe Commonwealth System of Higher Education; WO 01/123541 to Alexion,Inc.).

Various considerations can be involved in determining the extent ofhomology of target DNA sequences, such as, for example, the size of thetarget locus, availability of sequences, relative efficiency of doublecross-over events at the target locus and the similarity of the targetsequence with other sequences.

The targeting DNA can include a sequence in which DNA substantiallyisogenic flanks the desired sequence modifications with a correspondingtarget sequence in the genome to be modified. The substantially isogenicsequence can be at least about 95%, 97-98%, 99.0-99.5%, 99.6-99.9%, or100% identical to the corresponding target sequence (except for thedesired sequence modifications). The targeting DNA and the target DNApreferably can share stretches of DNA at least about 75, 150 or 500 basepairs that are 100% identical. Accordingly, targeting DNA can be derivedfrom cells closely related to the cell line being targeted; or thetargeting DNA can be derived from cells of the same cell line or animalas the cells being targeted.

The DNA constructs can be designed to modify the endogenous, targetalpha1,3GT. The homologous sequence for targeting the construct can haveone or more deletions, insertions, substitutions or combinationsthereof. The alteration can be the insertion of a selectable marker genefused in reading frame with the upstream sequence of the target gene.

Suitable selectable marker genes include, but are not limited to: genesconferring the ability to grow on certain media substrates, such as thetk gene (thymidine kinase) or the hprt gene (hypoxanthinephosphoribosyltransferase) which confer the ability to grow on HATmedium (hypoxanthine, aminopterin and thymidine); the bacterial gpt gene(guanine/xanthine phosphoribosyltransferase) which allows growth on MAXmedium (mycophenolic acid, adenine, and xanthine). See, for example,Song, K-Y., et al. Proc. Nat'l Acad. Sci. U.S.A. 84:6820-6824 (1987);Sambrook, J., et al., Molecular Cloning—A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1989), Chapter 16. Otherexamples of selectable markers include: genes conferring resistance tocompounds such as antibiotics, genes conferring the ability to grow onselected substrates, genes encoding proteins that produce detectablesignals such as luminescence, such as green fluorescent protein,enhanced green fluorescent protein (eGFP). A wide variety of suchmarkers are known and available, including, for example, antibioticresistance genes such as the neomycin resistance gene (neo) (Southern,P., and P. Berg, J. Mol. Appl. Genet. 1:327-341 (1982)); and thehygromycin resistance gene (hyg) (Nucleic Acids Research 11:6895-6911(1983), and Te Riele, H., et al., Nature 348:649-651 (1990)). Otherselectable marker genes include: acetohydroxyacid synthase (AHAS),alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase(GUS), chloramphenicol acetyltransferase (CAT), green fluorescentprotein (GFP), red fluorescent protein (RFP), yellow fluorescent protein(YFP), cyan fluorescent protein (CFP), horseradish peroxidase (HRP),luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), andderivatives thereof. Multiple selectable markers are available thatconfer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin,hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin,puromycin, and tetracycline.

Methods for the incorporation of antibiotic resistance genes andnegative selection factors will be familiar to those of ordinary skillin the art (see, e.g., WO 99/15650; U.S. Pat. No. 6,080,576; U.S. Pat.No. 6,136,566; Niwa et al., J. Biochem. 113:343-349 (1993); and Yoshidaet al., Transgenic Research 4:277-287 (1995)).

TABLE 1 Selectable marker genes that emit detectable signals U.S. Pat.No. Title 6,319,669 Modified green fluorescent proteins 6,316,181Establishment of cell lines with persistent expression of a greenfluorescent protein (GFP) using a pIRES/EGFP DNA vector construct6,303,373 Method of measuring plasma membrane targeting of GLUT46,291,177 Assay for agents which alter G-protein coupled receptoractivity 6,284,519 Cell systems having specific interaction of peptidebinding pairs 6,284,496 DNA vector for determining the presence ofout-of-reading-frame mutations 6,280,934 Assay for agents which alterG-protein coupled receptor activity 6,274,354 Methods using cre-lox forproduction of recombinant adeno-associated viruses 6,270,958 Detectionof negative-strand RNA viruses 6,268,201 IniB, iniA and iniC genes ofmycobacteria and methods of use 6,265,548 Mutant Aequorea victoriafluorescent proteins having increased cellular fluorescence 6,261,760Regulation of the cell cycle by sterols 6,255,558 Gene expression6,255,071 Mammalian viral vectors and their uses 6,251,677 Hybridadenovirus-AAV virus and methods of use thereof 6,251,602 Cell systemshaving specific interaction of peptide binding pairs 6,251,582Alternative G-coupled receptors associated with retroviral entry intocells, methods of identifying the same and diagnostic and therapeuticuses thereof 6,251,384 Metastasis models using green fluorescent protein(GFP) as a marker 6,248,558 Sequence and method for genetic engineeringof proteins with cell membrane translocating activity 6,248,550 Assaysfor protein kinases using fluorescent protein substrates 6,248,543Compositions and methods for screening antimicrobials 6,232,107Luciferases, fluorescent proteins, nucleic acids encoding theluciferases and fluorescent proteins and the use thereof in diagnostics,high throughput screening and novelty items 6,228,639 Vectors andmethods for the mutagenesis of mammalian genes 6,225,082 Myelin basicprotein MRNA transport and translation enhancer sequences 6,221,612Photon reducing agents for use in fluorescence assays 6,218,185 Piggybactransposon-based genetic transformation system for insects 6,214,567Immortalized human keratinocyte cell line 6,214,563 Photon reducingagents for reducing undesired light emission in assays 6,210,922 Serumfree production of recombinant proteins and adenoviral vectors 6,210,910Optical fiber biosensor array comprising cell populations confined tomicrocavities 6,203,986 Visualization of RNA in living cells 6,197,928Fluorescent protein sensors for detection of analytes 6,180,343 Greenfluorescent protein fusions with random peptides 6,172,188 Fluorescentproteins 6,153,409 Process for continuous optimized protein productionin insect larvae 6,150,176 Fluorescent protein sensors for measuring thepH of a biological sample 6,146,826 Green fluorescent protein 6,140,132Fluorescent protein sensors for measuring the pH of a biological sample6,136,539 Compositions and methods for the inhibition of MUC-5 mucingene expression 6,136,538 Silent inducible virus replicons and usesthereof 6,133,429 Chromophores useful for the preparation of noveltandem conjugates 6,130,313 Rapidly degrading GFP-fusion proteins6,124,128 Long wavelength engineered fluorescent proteins 6,110,711Method of defining cell types by probing comprehensive expressionlibraries with amplified RNA 6,096,865 Mutants of the green fluorescentprotein having improved fluorescent properties at 37 degrees 6,096,717Method for producing tagged genes transcripts and proteins 6,093,808 IκBeGFP constructs, cell lines and methods of use 6,090,919 FACS-optimizedmutants of the green fluorescent protein (GFP) 6,083,690 Methods andcompositions for identifying osteogenic agents 6,077,707 Long wavelengthengineered fluorescent proteins 6,066,476 Modified green fluorescentproteins 6,060,247 Post-mitotic neurons containing adenovirus vectorsthat modulate apoptosis and growth 6,054,321 Long wavelength engineeredfluorescent proteins 6,037,133 IκB eGFP constructs, cell lines andmethods of use 6,027,881 Mutant Aequorea victoria fluorescent proteinshaving increased cellular fluorescence 6,025,192 Modified retroviralvectors 6,020,192 Humanized green fluorescent protein genes and methods6,013,447 Random intracellular method for obtaining optimally activenucleic acid molecules 6,001,557 Adenovirus and methods of use thereof5,994,077 Fluorescence-based isolation of differentially induced genes5,994,071 Assessment of prostate cancer 5,993,778 Functional expressionof, and assay for, functional cellular receptors in vivo 5,989,808Identification of compounds affecting specific interaction of peptidebinding pairs 5,985,577 Protein conjugates containing multimers of greenfluorescent protein 5,968,773 System and method for regulation of geneexpression 5,968,738 Two-reporter FACS analysis of mammalian cells usinggreen fluorescent proteins 5,958,713 Method of detecting biologicallyactive substances by using green fluorescent protein 5,952,236Enzyme-based fluorescence biosensor for chemical analysis 5,948,889Compositions and methods for screening antimicrobials 5,948,681Non-viral vehicles for use in gene transfer 5,942,387 Combinatorialprocess for preparing substituted thiophene libraries 5,932,435Screening antisense and ribozyme nucleic acids in schizosaccharomycespombe 5,922,576 Simplified system for generating recombinantadenoviruses 5,919,445 Use of green fluorescent protein to trace theinfection of baculovirus in insects and to increase viral UV stability5,914,233 Screening assay for the identification of agents which alterexpression of PTH-rP

Combinations of selectable markers can also be used. For example, totarget alpha1,3GT, a neo gene (with or without its own promoter, asdiscussed above) can be cloned into a DNA sequence which is homologousto the alpha-1,3-GT gene. To use a combination of markers, the HSV-tkgene can be cloned such that it is outside of the targeting DNA (anotherselectable marker could be placed on the opposite flank, if desired).After introducing the DNA construct into the cells to be targeted, thecells can be selected on the appropriate antibiotics. In this particularexample, those cells which are resistant to G418 and gancyclovir aremost likely to have arisen by homologous recombination in which the neogene has been recombined into the alpha-1,3-GT gene but the tk gene hasbeen lost because it was located outside the region of the doublecrossover.

Deletions can be at least about 50 bp, more usually at least about 100bp, and generally not more than about 20 kbp, where the deletion cannormally include at least a portion of the coding region including aportion of or one or more exons, a portion of or one or more introns,and can or can not include a portion of the flanking non-coding regions,particularly the 5′-non-coding region (transcriptional regulatoryregion). Thus, the homologous region can extend beyond the coding regioninto the 5′-non-coding region or alternatively into the 3′-non-codingregion. Insertions can generally not exceed 10 kbp, usually not exceed 5kbp, generally being at least 50 bp, more usually at least 200 bp.

The region(s) of homology can include mutations, where mutations canfurther inactivate the target gene, in providing for a frame shift, orchanging a key amino acid, or the mutation can correct a dysfunctionalallele, etc. The mutation can be a subtle change, not exceeding about 5%of the homologous flanking sequences. Where mutation of a gene isdesired, the marker gene can be inserted into an intron or an exon.

The construct can be prepared in accordance with methods known in theart, various fragments can be brought together, introduced intoappropriate vectors, cloned, analyzed and then manipulated further untilthe desired construct has been achieved. Various modifications can bemade to the sequence, to allow for restriction analysis, excision,identification of probes, etc. Silent mutations can be introduced, asdesired. At various stages, restriction analysis, sequencing,amplification with the polymerase chain reaction, primer repair, invitro mutagenesis, etc. can be employed.

The construct can be prepared using a bacterial vector, including aprokaryotic replication system, e.g. an origin recognizable by E. coli,at each stage the construct can be cloned and analyzed. A marker, thesame as or different from the marker to be used for insertion, can beemployed, which can be removed prior to introduction into the targetcell. Once the vector containing the construct has been completed, itcan be further manipulated, such as by deletion of the bacterialsequences, linearization, introducing a short deletion in the homologoussequence. After final manipulation, the construct can be introduced intothe cell.

The present invention further includes recombinant constructs containingsequences of the alpha-1,3-GT gene. The constructs comprise a vector,such as a plasmid or viral vector, into which a sequence of theinvention has been inserted, in a forward or reverse orientation. Theconstruct can also include regulatory sequences, including, for example,a promoter, operably linked to the sequence. Large numbers of suitablevectors and promoters are known to those of skill in the art, and arecommercially available. The following vectors are provided by way ofexample. Bacterial: pBs, pQE-9 (Qiagen), phagescript, PsiX174,pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene);pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic:pWLneo, pSv2cat, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPv, pMSG, pSVL(Pharmiacia), viral origin vectors (M13 vectors, bacterial phage 1vectors, adenovirus vectors, and retrovirus vectors), high, low andadjustable copy number vectors, vectors which have compatible repliconsfor use in combination in a single host (pACYC184 and pBR322) andeukaryotic episomal replication vectors (pCDM8). Other vectors includeprokaryotic expression vectors such as pcDNA II, pSL301, pSE280, pSE380,pSE420, pTrcHisA, B, and C, pRSET A, B, and C (Invitrogen, Corp.),pGEMEX-1, and pGEMEX-2 (Promega, Inc.), the pET vectors (Novagen, Inc.),pTrc99 A, pKK223-3, the pGEX vectors, pEZZ18, pRIT2T, and pMC1871(Pharmacia, Inc.), pKK233-2 and pKK388-1 (Clontech, Inc.), and pProEx-HT(Invitrogen, Corp.) and variants and derivatives thereof. Other vectorsinclude eukaryotic expression vectors such as pFastBac, pFastBacHT,pFastBacDUAL, pSFV, and pTet-Splice (Invitrogen), pEUK-C1, pPUR, pMAM,pMAMneo, pBI101, pBI121, pDR2, pCMVEBNA, and pYACneo (Clontech), pSVK3,pSVL, pMSG, pCH110, and pKK232-8 (Pharmacia, Inc.), p3′SS, pXT1, pSG5,pPbac, pMbac, pMC1neo, and pOG44 (Stratagene, Inc.), and pYES2, pAC360,pBlueBacHis A, B, and C, pVL1392, pBlueBacIII, pCDM8, pcDNA1, pZeoSV,pcDNA3 pREP4, pCEP4, and pEBVHis (Invitrogen, Corp.) and variants orderivatives thereof. Additional vectors that can be used include: pUC18,pUC19, pBlueScript, pSPORT, cosmids, phagemids, YAC's (yeast artificialchromosomes), BAC's (bacterial artificial chromosomes), P1 (Escherichiacoli phage), pQE70, pQE60, pQE9 (quagan), pBS vectors, PhageScriptvectors, BlueScript vectors, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene),pcDNA3 (Invitrogen), pGEX, pTrsfus, pTrc99A, pET-5, pET-9, pKK223-3,pKK233-3, pDR540, pRIT5 (Pharmacia), pSPORT1, pSPORT2, pCMVSPORT2.0 andpSV-SPORT1 (Invitrogen), pTrxFus, pThioHis, pLEX, pTrcHis, pTrcHis2,pRSET, pBlueBacHis2, pcDNA3.1 /His, pcDNA3.1(−)/Myc-His, pSecTag,pEBVHis, pPIC9K, pPIC3.5K, pAO815, pPICZ, pPICZα, pGAPZ, pGAPZα,pBlueBac4.5, pBlueBacHis2, pMelBac, pSinRep5, pSinHis, pIND, pIND(SP1),pVgRXR, pcDNA2.1, pYES2, pZErO1.1, pZErO-2.1, pCR-Blunt, pSE280, pSE380,pSE420, pVL1392, pVL1393, pCDM8, pcDNA1.1, pcDNA1.1/Amp, pcDNA3.1,pcDNA3.1/Zeo, pSe, SV2, pRc/CMV2, pRc/RSV, pREP4, pREP7, pREP8, pREP9,pREP10, pCEP4, pEBVHis, pCR3.1, pCR2.1, pCR3.1-Uni, and pCRBac fromInvitrogen; λExCell, λgt11, pTrc99A, pKK223-3, pGEX-10T, pGEX-2T,pGEX-2TK, pGEX-4T-1, pGEX-4T-2, pGEX-4T-3, pGEX-3X, pGEX-5x-1,pGEX-5x-2, pGEX-5x-3, pEZZ18, pRIT2T, pMC1871, pSVK3, pSVL, pMSG,pCH110, pKK232-8, pSL1180, pNEO, and pUC4K from Pharmacia;pSCREEN-1b(+), pT7Blue(R), pT7Blue-2, pCITE-4abc(+), pOCUS-2, pTAg,pET-32LIC, pET-30LIC, pBAC-2cp LIC, pBACgus-2cp LIC, pT7Blue-2LIC,pT7Blue-2, λSCREEN-1, λBlueSTAR, pET-3abcd, pET-7abc, pET9abcd,pET11abcd, pET12abc, pET-14b, pET-15b, pET-16b, pET-17b-pET-17xb,pET-19b, pET-20b(+), pET-21abcd(+), pET-22b(+), pET-23abcd(+),pET-24abcd(+), pET-25 b(+), pET-26b(+), pET-27b(+), pET-28 abc(+),pET-29abc(+), pET-30 abc(+), pET-31b(+), pET-32abc(+), pET-33b(+),pBAC-1, pBACgus-1, pBAC4x-1, pBACgus4x-1, pBAC-3cp, pBACgus-2cp,pBACsurf-1, plg, Signal plg, pYX, Selecta Vecta-Neo, Selecta Vecta-Hyg,and Selecta Vecta-Gpt from Novagen; pLexA, pB42AD, pGBT9, pAS2-1,pGAD424, pACT2, pGAD GL, pGAD GH, pGAD10, pGilda, pEZM3, pEGFP, pEGFP-1,pEGFP-N, pEGFP-C, pEBFP, pGFPuv, pGFP, p6xHis-GFP, pSEAP2-Basic,pSEAP2-Contral, pSEAP2-Promoter, pSEAP2-Enhancer, pβgal-Basic,pβgal-Control, pβgal-Promoter, βgal-Enhancer, pCMVβ, pTet-Off, pTet-On,pTK-Hyg, pRetro-Off, pRetro-On, pIRES1neo, pIRES1hyg, pLXSN, pLNCX,pLAPSN, pMAMneo, pMAMneo-CAT, pMAMneo-LUC, pPUR, pSV2neo, pYEX4T-1/2/3,pYEX-S1, pBacPAK-His, pBacPAK8/9, pAcUW31, BacPAK6, pTriplEx, λgt10,λgt11, pWE15, and λTriplEx from Clontech; Lambda ZAP II, pBK-CMV,pBK-RSV, pBluescript II KS+/−, pBluescript II SK+/−, pAD-GAL4, pBD-GAL4Cam, pSurfscript, Lambda FIX II, Lambda DASH, Lambda EMBL3, LambdaEMBL4, SuperCos, pCR-Script Amp, pCR-Script Cam, pCR-Script Direct, pBS+/−, pBC KS+/−, pBC SK+/−, Phagescript, pCAL-n-EK, pCAL-n, pCAL-c,pCAL-kc, pET-3abcd, pET-11abcd, pSPUTK, pESP-1, pCMVLacI, pOPRSVI/MCS,pOPI3 CAT, pXT1, pSG5, pPbac, pMbac, pMC1neo, pMC1neo Poly A, pOG44,pOG45, pFRTβGAL, pNEOβGAL, pRS403, pRS404, pRS405, pRS406, pRS413,pRS414, pRS415, and pRS416 from Stratagene and variants or derivativesthereof. Two-hybrid and reverse two-hybrid vectors can also be used, forexample, pPC86, pDBLeu, pDBTrp, pPC97, p2.5, pGADI-3, pGAD10, pACt,pACT2, pGADGL, pGADGH, pAS2-1, pGAD424, pGBT8, pGBT9, pGAD-GAL4, pLexA,pBD-GAL4, pHISi, pHISi-1, placZi, pB42AD, pDG202, pJK202, pJG4-5,pNLexA, pYESTrp and variants or derivatives thereof. Any other plasmidsand vectors may be used as long as they are replicable and viable in thehost.

Techniques which can be used to allow the DNA construct entry into thehost cell include calcium phosphate/DNA co precipitation, microinjectionof DNA into the nucleus, electroporation, bacterial protoplast fusionwith intact cells, transfection, or any other technique known by oneskilled in the art. The DNA can be single or double stranded, linear orcircular, relaxed or supercoiled DNA. For various techniques fortransfecting mammalian cells, see, for example, Keown et al., Methods inEnzymology Vol. 185, pp. 527-537 (1990).

In one specific embodiment, heterozygous knockout cells can be producedby transfection of primary porcine fetal fibroblasts with a knockoutvector containing alpha-1,3-GT sequence isolated from isogenic DNA. Asdescribed in Dai et al. (Nature Biotchnology, 20:451-455), the 5′ armcan be 4.9 kb and be comprised of a large fragment of intron 8 and the5′ end of exon 9. The 3′ arm can be and be comprised of exon 9 sequence.The vector can incorporate a promoter trap strategy, using, for example,IRES (internal ribosome entry site) to initiate translation of the Neorgene (see, for example, FIG. 6).

Selection of Homologously Recombined Cells

The cells can then be grown in appropriately-selected medium to identifycells providing the appropriate integration. The presence of theselectable marker gene inserted into the alpha-1,3-GT gene establishesthe integration of the target construct into the host genome. Thosecells which show the desired phenotype can then be further analyzed byrestriction analysis, electrophoresis, Southern analysis, polymerasechain reaction, etc to analyze the DNA in order to establish whetherhomologous or non-homologous recombination occurred. This can bedetermined by employing probes for the insert and then sequencing the 5′and 3′ regions flanking the insert for the presence of the alpha-1,3-GTgene extending beyond the flanking regions of the construct oridentifying the presence of a deletion, when such deletion isintroduced. Primers can also be used which are complementary to asequence within the construct and complementary to a sequence outsidethe construct and at the target locus. In this way, one can only obtainDNA duplexes having both of the primers present in the complementarychains if homologous recombination has occurred. By demonstrating thepresence of the primer sequences or the expected size sequence, theoccurrence of homologous recombination is supported.

The polymerase chain reaction used for screening homologousrecombination events is known in the art, see, for example, Kim andSmithies, Nucleic Acids Res. 16:8887-8903, 1988; and Joyner et al.,Nature 338:153-156, 1989. The specific combination of a mutant polyomaenhancer and a thymidine kinase promoter to drive the neomycin gene hasbeen shown to be active in both embryonic stem cells and EC cells byThomas and Capecchi, supra, 1987; Nicholas and Berg (1983) inTeratocarcinoma Stem Cell, eds. Siver, Martin and Strikland (Cold SpringHarbor Lab., Cold Spring Harbor, N.Y. (pp. 469-497); and Linney andDonerly, Cell 35:693-699, 1983.

The cell lines obtained from the first round of targeting are likely tobe heterozygous for the targeted allele. Homozygosity, in which bothalleles are modified, can be achieved in a number of ways. One approachis to grow up a number of cells in which one copy has been modified andthen to subject these cells to another round of targeting using adifferent selectable marker. Alternatively, homozygotes can be obtainedby breeding animals heterozygous for the modified allele, according totraditional Mendelian genetics. In some situations, it can be desirableto have two different modified alleles. This can be achieved bysuccessive rounds of gene targeting or by breeding heterozygotes, eachof which carries one of the desired modified alleles.

Induced Mutation in the Alpha 1,3 GT Locus

In certain other embodiments, the methods of the invention involve theintentional introduction of a mutation via a mutagenic agent. Examplesof mutagenic agents known in the art and suitable for use in the presentinvention include, but are not limited to, chemical mutagens (e.g.,DNA-intercalating or DNA-binding chemicals such as N-ethyl-N-nitrosourea(ENU), ethylmethanesulphonate (EMS), mustard gas, ICR191 and the like;see, e.g., E. C. Friedberg, G. C. Walker, W. Siede, DNA Repair andMutagenesis, ASM Press, Washington D.C. (1995), physical mutagens (e.g.,UV radiation, radiation, x-rays), biochemical mutagens (e.g.,restriction enzymes, DNA repair mutagens, DNA repair inhibitors, anderror-prone DNA polymerases and replication proteins), as well astransposon insertion. According to the methods of the present invention,cells in culture can be exposed to one of these agents, and any mutationresulting in the depletion of galactose alpha1,3-galactose on the cellsurface can be selected, for example, via exposure to toxin A.

Preferred doses of chemical mutagens for inducing mutations in cells areknown in the art, or can be readily determined by the ordinarily skilledartisan using assays of mutagenesis known in the art. Chemicalmutagenesis of cells in vitro can be achieved by treating the cells withvarious doses of the mutagenic agent and/or controlling the time ofexposure to the agent. By titrating the mutagenic agent exposure and/ordose, it is possible to carry out the optimal degree of mutagenesis forthe intended purpose, thereby mutating a desired number of genes in eachtarget cell. For example, useful doses of ENU can be 0.1-0.4 mg/ml forapproximately 1-2 hours. In another example, useful doses of EMS can be0.1-1 mg/ml for approximately 10-30 hours. In addition, lower and higherdoses and exposure times can also be used to achieve the desiredmutation frequency.

II. Identification of Cells that do Not Express Functional Alpha-1,3-GT

In another aspect of the present invention, a selection method isprovided for determining whether porcine cells lack expression offunctional alpha-1,3-GT.

In one embodiment, the selection procedure can be based on a bacterialtoxin to select for cells that lack expression of functional alpha1,3GT.In another embodiment, the bacterial toxin, toxin A produced byClostridium difficile, can be used to select for cells lacking the cellsurface epitope galactose alpha1,3-galactose. Exposure to C. difficiletoxin can cause rounding of cells that exhibit this epitope on theirsurface, releasing the cells from the plate matrix. Both targeted geneknockouts and mutations that disable enzyme function or expression canbe detected using this selection method. Cells lacking cell surfaceexpression of the galactose alpha 1,3-galactose epitope, identifiedusing Toxin A mediated selection described, or produced using standardmethods of gene inactivation including gene targeting, can then be usedto produce pigs, in which both alleles of the alpha 1,3 GT gene areinactive.

In one embodiment, the selection method can detect the depletion of thealpha 1,3GT epitope directly, whether due to targeted knockout of thealpha 1,3GT gene by homologous recombination, or a mutation in the genethat results in a nonfunctioning or nonexpressed enzyme. Selection viaantibiotic resistance has been used most commonly for screening (seeabove). This method can detect the presence of the resistance gene onthe targeting vector, but does not directly indicate whether integrationwas a targeted recombination event or a random integration. Certaintechnology, such as Poly A and promoter trap technology, increase theprobability of targeted events, but again, do not give direct evidencethat the desired phenotype, a cell deficient in gal alpha 1,3 galepitopes on the cell surface, has been achieved. In addition, negativeforms of selection can be used to select for targeted integration; inthese cases, the gene for a factor lethal to the cells is inserted insuch a way that only targeted events allow the cell to avoid death.Cells selected by these methods can then be assayed for gene disruption,vector integration and, finally, alpha 1,3gal epitope depletion. Inthese cases, since the selection is based on detection of targetingvector integration and not at the altered phenotype, only targetedknockouts, not point mutations, gene rearrangements or truncations orother such modifications can be detected.

Toxin A, a cytotoxin produced by the bacterium Clostridium difficile,specifically binds the terminal carbohydrate glactose alpha1,3-galactosesequence gal alpha 1-3gal beta 1-4GlcNAc. Binding to this receptormediates a cytotoxic effect on the cell, causing it to change morphologyand, in some cases, to release from the plate matrix. Under controlledconditions, cells not carrying this marker are unaffected by the toxin.Thus, in one embodiment, to determine whether or not the alpha 1,3 galepitope has been successfully eliminated via targeted knockout or genemutation of the gal alpha-1,3-GT locus, cells that do not carry theepitope can be selected. Exposure to toxin A can be toxic for cellscarrying the epitope, and promote selection for those cells in which thegene has been successfully inactivated. Thus, according to on aspect ofthe present invention, cells useful as nuclear donors for production ofgenetically altered animals (e.g., pigs) that are knocked out or mutatedin the gal alpha 1,3 locus are selected by exposure of cells to C.difficile toxin A.

Toxin A, one of two cytotoxins produced by Clostridium difficile, has ahigh binding affinity for the galactose alpha1,3-galactose sequence galalpha 1,3-gal beta 1,4GlcNAc found on the surface of a variety of celltypes (Clark et al., Arch. Biochem. Biophys. 257 (1): 217-229, 1987).This carbohydrate seems to serve as a functional receptor for Toxin A,as cells displaying this epitope on their surface are more sensitive tothe cytotoxic effect of toxin A than are cells lacking this receptor.Sensitive cells exposed to toxin A in culture exhibit cell rounding,probably due to actin depolymerization and resultant changes incytoskeletal integrity (Kushnaryov et al., J. Biol. Chem. 263:17755-17762 (1988) and Just et al., J. Clin. Invest. 95:1026-1031,1995). These cells can be selectively removed from theculture, as they lift from the matrix and float in suspension, leavingunaffected cells firmly attached to the plate surface.

Exposure of cells to toxin A. In one embodiment, attached cells areexposed to toxin A as a component of cell culture media. After a fixedtime of exposure, the media containing the toxin A and released toxinA-sensitive cells are removed, the plate washed, and the media, withouttoxin A, replenished. The exposure to toxin A is repeated over a periodof days to remove attached toxin-sensitive cells from the plates, andallow insensitive cells to proliferate and expand. Purified toxin A canbe used in the methods of the present invention (available commercially,see for example, Techlab Inc., Cat.# T3001, Blacksburg, Va.). Crudeunpurified toxin A can also be used (available commercially, see forexample, Techlab Inc. Cat. #T5000 or T3000, Blacksburg, Va.), which canrequire initial titering to determine effective dosage for selection.

Serum-Based Selection Method

In another embodiment, the selection procedure can be conducted usingserum containing complement factors and natural antibodies to the galalpha1,3gal epitope (see, for example, Koike et al., Xenotransplantation4:147-153, 1997). Exposure to serum from a human or non-human primatethat contains anti-Gal antibodies can cause cell lysis due to specificantibody binding and complement activation in cells that exhibit galalpha 1,3 gal epitope. Therefore, cells deficient in alpha-1,3-GT willremain alive and thus can be selected.

Further Characterization of Porcine Cells Lacking Expression ofFunctional alpha1,3GT

Porcine cells believed to lacking expression of functional alpha-1,3-GTcan be further characterized. Such characterization can be accomplishedby the following techniques, including, but not limited to: PCRanalysis, Southern blot analysis, Northern blot analysis, specificlectin binding assays, and/or sequencing analysis.

PCR analysis as described in the art (see, for example, Dai et al.Nature Biotechnology 20:431-455) can be used to determine theintegration of targeting vectors. In one embodiment, amplimers canoriginate in the antibiotic resistance gene and extend into a regionoutside the vector sequence. Southern analysis (see, for example, Dai etal. Nature Biotechnology 20:431-455) can also be used to characterizegross modifications in the locus, such as the integration of a targetingvector into the alpha 1,3GT locus. Whereas, Northern analysis can beused to characterize the transcript produced from each of the alleles.

Specific lectin binding, using GSL IB4 lectin from Griffonia(Bandeiraea) simplicifolia (Vector Labs), a lectin that specificallybinds the carbohydrate moiety gal alpha 1,3 gal, and FACS (fluorescentantibody cell sorting) analysis of binding can determine whether or notthe alpha 1,3 gal epitope is present on the cells. This type of analysisinvolves the addition of fluorescein-labeled GSL-IB4 lectin to the cellsand subsequent cell sorting.

Further, sequencing analysis of the cDNA produced from the RNAtranscript can also be used to determine the precise location of anymutations in the alpha 1,3GT allele.

III. Production of Porcine Animals

In yet another aspect, the present invention provides a method forproducing viable pigs in which both alleles of the alpha-1,3-GT genehave been rendered inactive. In one embodiment, the pigs are produced bycloning using a donor nucleus from a porcine cell in which both allelesof the alpha-1,3-GT gene have been inactivated. In one embodiment, bothalleles of the alpha-1,3-GT gene are inactivated via a genetic targetingevent. In another embodiment, both alleles of the alpha-1,3-GT gene areinactivated due to the presence of a point mutation. In anotherembodiment, one allele is inactivated by a genetic targeting event andthe other allele is inactivated via a point mutation. In a furtherembodiment, one allele is inactivated by a genetic targeting event andthe other allele is inactivated due to presence of a T-to-G pointmutation at the second base of exon 9 of the alpha-1,3-GT gene. In aspecific embodiment, one allele is inactivated via a targeting constructdirected to Exon 9 (FIG. 6) and the other allele is inactivated due topresence of a T-to-G point mutation at the second base of exon 9 of thealpha-1,3-GT gene. In another embodiment, a method to clone such pigsincludes: enucleating an oocyte, fusing the oocyte with a donor nucleusfrom a porcine cell in which both alleles of the alpha-1,3-GT gene havebeen inactivated, and implanting the nuclear transfer-derived embryointo a surrogate mother.

Alternatively, a method is provided for producing viable pigs that lackany expression of functional alpha-1,3-GT by inactivating both allelesof the alpha-1,3-GT gene in embryonic stem cells, which can then be usedto produce offspring.

Genetically altered animals that can be created by modifying zygotesdirectly. For mammals, the modified zygotes can be then introduced intothe uterus of a pseudopregnant female capable of carrying the animal toterm. For example, if whole animals lacking the alpha-1,3-GT gene aredesired, then embryonic stem cells derived from that animal can betargeted and later introduced into blastocysts for growing the modifiedcells into chimeric animals. For embryonic stem cells, either anembryonic stem cell line or freshly obtained stem cells can be used.

In a suitable embodiment of the invention, the totipotent cells areembryonic stem (ES) cells. The isolation of ES cells from blastocysts,the establishing of ES cell lines and their subsequent cultivation arecarried out by conventional methods as described, for example, byDoetchmann et al., J. Embryol. Exp. Morph. 87:27-45 (1985); Li et al.,Cell 69:915-926 (1992); Robertson, E. J. “Tetracarcinomas and EmbryonicStem Cells: A Practical Approach,” ed. E. J. Robertson, IRL Press,Oxford, England (1987); Wurst and Joyner, “Gene Targeting: A PracticalApproach,” ed. A. L. Joyner, IRL Press, Oxford, England (1993); Hogen etal., “Manipulating the Mouse Embryo: A Laboratory Manual,” eds. Hogan,Beddington, Costantini and Lacy, Cold Spring Harbor Laboratory Press,New York (1994); and Wang et al., Nature 336:741-744 (1992). In anothersuitable embodiment of the invention, the totipotent cells are embryonicgerm (EG) cells. Embryonic Germ cells are undifferentiated cellsfunctionally equivalent to ES cells, that is they can be cultured andtransfected in vitro, then contribute to somatic and germ cell lineagesof a chimera (Stewart et al., Dev. Biol. 161:626-628 (1994)). EG cellsare derived by culture of primordial germ cells, the progenitors of thegametes, with a combination of growth factors: leukemia inhibitoryfactor, steel factor and basic fibroblast growth factor (Matsui et al.,Cell 70:841-847 (1992); Resnick et al., Nature 359:550-551 (1992)). Thecultivation of EG cells can be carried out using methods described inthe article by Donovan et al., “Transgenic Animals, Generation and Use,”Ed. L. M. Houdebine, Harwood Academic Publishers (1997), and in theoriginal literature cited therein.

Tetraploid blastocysts for use in the invention may be obtained bynatural zygote production and development, or by known methods byelectrofusion of two-cell embryos and subsequently cultured asdescribed, for example, by James et al., Genet. Res. Camb. 60:185-194(1992); Nagy and Rossant, “Gene Targeting: A Practical Approach,” ed. A.L. Joyner, IRL Press, Oxford, England (1993); or by Kubiak andTarkowski, Exp. Cell Res. 157:561-566 (1985).

The introduction of the ES cells or EG cells into the blastocysts can becarried out by any method known in the art. A suitable method for thepurposes of the present invention is the microinjection method asdescribed by Wang et al., EMBO J. 10:2437-2450 (1991).

Alternatively, by modified embryonic stem cells transgenic animals canbe produced. The genetically modified embryonic stem cells can beinjected into a blastocyst and then brought to term in a female hostmammal in accordance with conventional techniques. Heterozygous progenycan then be screened for the presence of the alteration at the site ofthe target locus, using techniques such as PCR or Southern blotting.After mating with a wild-type host of the same species, the resultingchimeric progeny can then be cross-mated to achieve homozygous hosts.

After transforming embryonic stem cells with the targeting vector toalter the alpha-1,3-GT gene, the cells can be plated onto a feeder layerin an appropriate medium, e.g., fetal bovine serum enhanced DMEM. Cellscontaining the construct can be detected by employing a selectivemedium, and after sufficient time for colonies to grow, colonies can bepicked and analyzed for the occurrence of homologous recombination.Polymerase chain reaction can be used, with primers within and withoutthe construct sequence but at the target locus. Those colonies whichshow homologous recombination can then be used for embryo manipulatingand blastocyst injection. Blastocysts can be obtained from superovulatedfemales. The embryonic stem cells can then be trypsinized and themodified cells added to a droplet containing the blastocysts. At leastone of the modified embryonic stem cells can be injected into theblastocoel of the blastocyst. After injection, at least one of theblastocysts can be returned to each uterine horn of pseudopregnantfemales. Females are then allowed to go to term and the resultinglitters screened for mutant cells having the construct. The blastocystsare selected for different parentage from the transformed ES cells. Byproviding for a different phenotype of the blastocyst and the ES cells,chimeric progeny can be readily detected, and then genotyping can beconducted to probe for the presence of the modified alpha-1,3-GT gene.

Somatic Cell Nuclear Transfer to Produce Cloned, Transgenic Offspring

The present invention provides a method for cloning a pig lacking afunctional alpha-1,3-GT gene via somatic cell nuclear transfer. Ingeneral, the pig can be produced by a nuclear transfer processcomprising the following steps: obtaining desired differentiated pigcells to be used as a source of donor nuclei; obtaining oocytes from apig; enucleating said oocytes; transferring the desired differentiatedcell or cell nucleus into the enucleated oocyte, e.g., by fusion orinjection, to form NT units; activating the resultant NT unit; andtransferring said cultured NT unit to a host pig such that the NT unitdevelops into a fetus.

Nuclear transfer techniques or nuclear transplantation techniques areknown in the art(Dai et al. Nature Biotechnology 20:251-255; Polejaevaet al Nature 407:86-90 (2000); Campbell et al, Theriogenology, 43:181(1995); Collas et al, Mol. Report Dev., 38:264-267 (1994); Keefer et al,Biol. Reprod., 50:935-939 (1994); Sims et al, Proc. Natl. Acad. Sci.,USA, 90:6143-6147 (1993); WO 94/26884; WO 94/24274, and WO 90/03432,U.S. Pat. Nos. 4,944,384 and 5,057,420).

A donor cell nucleus, which has been modified to alter the alpha-1,3-GTgene, is transferred to a recipient porcine oocyte. The use of thismethod is not restricted to a particular donor cell type. The donor cellcan be as described herein, see also, for example, Wilmut et al Nature385 810 (1997); Campbell et al Nature 380 64-66 (1996); Dai et al.,Nature Biotechnology 20:251-255, 2002 or Cibelli et al Science 2801256-1258 (1998). All cells of normal karyotype, including embryonic,fetal and adult somatic cells which can be used successfully in nucleartransfer can be employed. Fetal fibroblasts are a particularly usefulclass of donor cells. Generally suitable methods of nuclear transfer aredescribed in Campbell et al Theriogenology 43 181 (1995), Dai et al.Nature Biotechnology 20:251-255, Polejaeva et al Nature 407:86-90(2000), Collas et al Mol. Reprod. Dev. 38 264-267 (1994), Keefer et alBiol. Reprod. 50 935-939 (1994), Sims et al Proc. Nat'l. Acad. Sci. USA90 6143-6147 (1993), WO-A-9426884, WO-A-9424274, WO-A-9807841,WO-A-9003432, U.S. Pat. No. 4,994,384 and U.S. Pat. No. 5,057,420.Differentiated or at least partially differentiated donor cells can alsobe used. Donor cells can also be, but do not have to be, in culture andcan be quiescent. Nuclear donor cells which are quiescent are cellswhich can be induced to enter quiescence or exist in a quiescent statein vivo. Prior art methods have also used embryonic cell types incloning procedures (Campbell et al (Nature, 380:64-68, 1996) and Sticeet al (Biol. Reprod., 20 54:100-110, 1996).

Somatic nuclear donor cells may be obtained from a variety of differentorgans and tissues such as, but not limited to, skin, mesenchyme, lung,pancreas, heart, intestine, stomach, bladder, blood vessels, kidney,urethra, reproductive organs, and a disaggregated preparation of a wholeor part of an embryo, fetus, or adult animal. In a suitable embodimentof the invention, nuclear donor cells are selected from the groupconsisting of epithelial cells, fibroblast cells, neural cells,keratinocytes, hematopoietic cells, melanocytes, chondrocytes,lymphocytes (B and T), macrophages, monocytes, mononuclear cells,cardiac muscle cells, other muscle cells, granulosa cells, cumuluscells, epidermal cells or endothelial cells. In another embodiment, thenuclear donor cell is an embryonic stem cell. In a preferred embodiment,fibroblast cells can be used as donor cells.

In another embodiment of the invention, the nuclear donor cells of theinvention are germ cells of an animal. Any germ cell of an animalspecies in the embryonic, fetal, or adult stage may be used as a nucleardonor cell. In a suitable embodiment, the nuclear donor cell is anembryonic germ cell.

Nuclear donor cells may be arrested in any phase of the cell cycle (G0,G1, G2, S, M) so as to ensure coordination with the acceptor cell. Anymethod known in the art may be used to manipulate the cell cycle phase.Methods to control the cell cycle phase include, but are not limited to,G0 quiescence induced by contact inhibition of cultured cells, G0quiescence induced by removal of serum or other essential nutrient, G0quiescence induced by senescence, G0 quiescence induced by addition of aspecific growth factor; G0 or G1 quiescence induced by physical orchemical means such as heat shock, hyperbaric pressure or othertreatment with a chemical, hormone, growth factor or other substance;S-phase control via treatment with a chemical agent which interfereswith any point of the replication procedure; M-phase control viaselection using fluorescence activated cell sorting, mitotic shake off,treatment with microtubule disrupting agents or any chemical whichdisrupts progression in mitosis (see also Freshney, R. I., “Culture ofAnimal Cells: A Manual of Basic Technique,” Alan R. Liss, Inc, New York(1983).

Methods for isolation of oocytes are well known in the art. Essentially,this can comprise isolating oocytes from the ovaries or reproductivetract of a pig. A readily available source of pig oocytes isslaughterhouse materials. For the combination of techniques such asgenetic engineering, nuclear transfer and cloning, oocytes mustgenerally be matured in vitro before these cells can be used asrecipient cells for nuclear transfer, and before they can be fertilizedby the sperm cell to develop into an embryo. This process generallyrequires collecting immature (prophase I) oocytes from mammalianovaries, e.g., bovine ovaries obtained at a slaughterhouse, and maturingthe oocytes in a maturation medium prior to fertilization or enucleationuntil the oocyte attains the metaphase II stage, which in the case ofbovine oocytes generally occurs about 18-24 hours post-aspiration. Thisperiod of time is known as the “maturation period”. In certainembodiments, the oocyte is obtained from a gilt. A “gilt” is a femalepig that has never had offspring. In other embodiments, the oocyte isobtained from a sow. A “sow” is a female pig that has previouslyproduced offspring.

A metaphase II stage oocyte can be the recipient oocyte, at this stageit is believed that the oocyte can be or is sufficiently “activated” totreat the introduced nucleus as it does a fertilizing sperm. MetaphaseII stage oocytes, which have been matured in vivo have been successfullyused in nuclear transfer techniques. Essentially, mature metaphase IIoocytes can be collected surgically from either non-superovulated orsuperovulated porcine 35 to 48, or 39-41, hours past the onset of estrusor past the injection of human chorionic gonadotropin (hCG) or similarhormone.

After a fixed time maturation period, which ranges from about 10 to 40hours, and preferably about 16-18 hours, the oocytes can be enucleated.Prior to enucleation the oocytes can be removed and placed inappropriate medium, such as HECM containing 1 milligram per milliliterof hyaluronidase prior to removal of cumulus cells. The stripped oocytescan then be screened for polar bodies, and the selected metaphase IIoocytes, as determined by the presence of polar bodies, are then usedfor nuclear transfer. Enucleation follows.

Enucleation can be performed by known methods, such as described in U.S.Pat. No. 4,994,384. For example, metaphase II oocytes can be placed ineither HECM, optionally containing 7.5 micrograms per millilitercytochalasin B, for immediate enucleation, or can be placed in asuitable medium, for example an embryo culture medium such as CR1aa,plus 10% estrus cow serum, and then enucleated later, preferably notmore than 24 hours later, and more preferably 16-18 hours later.

Enucleation can be accomplished microsurgically using a micropipette toremove the polar body and the adjacent cytoplasm. The oocytes can thenbe screened to identify those of which have been successfullyenucleated. One way to screen the oocytes is to stain the oocytes with 1microgram per milliliter 33342 Hoechst dye in HECM, and then view theoocytes under ultraviolet irradiation for less than 10 seconds. Theoocytes that have been successfully enucleated can then be placed in asuitable culture medium, for example, CR1aa plus 10% serum.

A single mammalian cell of the same species as the enucleated oocyte canthen be transferred into the perivitelline space of the enucleatedoocyte used to produce the NT unit. The mammalian cell and theenucleated oocyte can be used to produce NT units according to methodsknown in the art. For example, the cells can be fused by electrofusion.Electrofusion is accomplished by providing a pulse of electricity thatis sufficient to cause a transient breakdown of the plasma membrane.This breakdown of the plasma membrane is very short because the membranereforms rapidly. Thus, if two adjacent membranes are induced tobreakdown and upon reformation the lipid bilayers intermingle, smallchannels can open between the two cells. Due to the thermodynamicinstability of such a small opening, it enlarges until the two cellsbecome one. See, for example, U.S. Pat. No. 4,997,384 by Prather et al.A variety of electrofusion media can be used including, for example,sucrose, mannitol, sorbitol and phosphate buffered solution. Fusion canalso be accomplished using Sendai virus as a fusogenic agent (Graham,Wister Inot. Symp. Monogr., 9, 19, 1969). Also, the nucleus can beinjected directly into the oocyte rather than using electroporationfusion. See, for example, Collas and Barnes, Mol. Reprod. Dev.,38:264-267 (1994). After fusion, the resultant fused NT units are thenplaced in a suitable medium until activation, for example, CR1aa medium.Typically activation can be effected shortly thereafter, for exampleless than 24 hours later, or about 4-9 hours later, or optimally 1-2hours after fusion. In a preferred embodiments, activation occurs atleast one hour post fusion and at 40-41 hours post maturation.

The NT unit can be activated by known methods. Such methods include, forexample, culturing the NT unit at sub-physiological temperature, inessence by applying a cold, or actually cool temperature shock to the NTunit. This can be most conveniently done by culturing the NT unit atroom temperature, which is cold relative to the physiologicaltemperature conditions to which embryos are normally exposed.Alternatively, activation can be achieved by application of knownactivation agents. For example, penetration of oocytes by sperm duringfertilization has been shown to activate prefusion oocytes to yieldgreater numbers of viable pregnancies and multiple genetically identicalcalves after nuclear transfer. Also, treatments such as electrical andchemical shock can be used to activate NT embryos after fusion. See, forexample, U.S. Pat. No. 5,496,720, to Susko-Parrish et al. Additionally,activation can be effected by simultaneously or sequentially byincreasing levels of divalent cations in the oocyte, and reducingphosphorylation of cellular proteins in the oocyte. This can generallybe effected by introducing divalent cations into the oocyte cytoplasm,e.g., magnesium, strontium, barium or calcium, e.g., in the form of anionophore. Other methods of increasing divalent cation levels includethe use of electric shock, treatment with ethanol and treatment withcaged chelators. Phosphorylation can be reduced by known methods, forexample, by the addition of kinase inhibitors, e.g., serine-threoninekinase inhibitors, such as 6-dimethyl-aminopurine, staurosporine,2-aminopurine, and sphingosine. Alternatively, phosphorylation ofcellular proteins can be inhibited by introduction of a phosphatase intothe oocyte, e.g., phosphatase 2A and phosphatase 2B.

The activated NT units, or “fused embyos”, can then be cultured in asuitable in vitro culture medium until the generation of cell colonies.Culture media suitable for culturing and maturation of embryos are wellknown in the art. Examples of known media, which can be used for embryoculture and maintenance, include Ham's F-10+10% fetal calf serum (FCS),Tissue Culture Medium-199 (TCM-199)+10% fetal calf serum,Tyrodes-Albumin-Lactate-Pyruvate (TALP), Dulbecco's Phosphate BufferedSaline (PBS), Eagle's and Whitten's media, and, in one specific example,the activated NT units can be cultured in NCSU-23 medium for about 1-4 hat approximately 38.6° C. in a humidified atmosphere of 5% CO2.

Afterward, the cultured NT unit or units can be washed and then placedin a suitable media contained in well plates which preferably contain asuitable confluent feeder layer. Suitable feeder layers include, by wayof example, fibroblasts and epithelial cells. The NT units are culturedon the feeder layer until the NT units reach a size suitable fortransferring to a recipient female, or for obtaining cells which can beused to produce cell colonies. Preferably, these NT units can becultured until at least about 2 to 400 cells, about 4 to 128 cells, orat least about 50 cells.

Activated NT units can then be transferred (embryo transfers) to theoviduct of an female pigs. In one embodiment, the female pigs can be anestrus-synchronized recipient gilt. Crossbred gilts (largewhite/Duroc/Landrace) (280-400 lbs) can be used. The gilts can besynchronized as recipient animals by oral administration of 18-20 mgRegu-Mate (Altrenogest, Hoechst, Warren, N.J.) mixed into the feed.Regu-Mate can be fed for 14 consecutive days. One thousand units ofHuman Chorionic Gonadotropin (hCG, Intervet America, Millsboro, Del.)can then be administered i.m. about 105 h after the last Regu-Matetreatment. Embryo transfers of the can then be performed about 22-26 hafter the hCG injection. In one embodiment, the pregnancy can be broughtto term and result in the birth of live offspring. In anotherembodiment, the pregnancy can be terminated early and embryonic cellscan be harvested.

Breeding for Desired Homozygous Knockout Animals

In another aspect, the present invention provides a method for producingviable pigs that lack any expression of functional alpha-1,3-GT isprovided by breeding a male pig heterozygous for the alpha-1,3-GT genewith a female pig heterozygous for the alpha-1,3-GT gene. In oneembodiment, the pigs are heterozygous due to the genetic modification ofone allele of the alpha-1,3-GT gene to prevent expression of thatallele. In another embodiment, the pigs are heterozygous due to thepresence of a point mutation in one allele of the alpha-1,3-GT gene. Inanother embodiment, the point mutation can be a T-to-G point mutation atthe second base of exon 9 of the alpha-1,3-GT gene. In one specificembodiment, a method to produce a porcine animal that lacks anyexpression of functional alpha-1,3-GT is provided wherein a male pigthat contains a T-to-G point mutation at the second base of exon 9 ofthe alpha-1,3-GT gene is bred with a female pig that contains a T-to-Gpoint mutation at the second base of exon 9 of the alpha-1,3-GT gene.

In one embodiment, sexually mature animals produced from nucleartransfer from donor cells that carrying a double knockout in thealpha-1,3-GT gene, can be bred and their offspring tested for thehomozygous knockout. These homozygous knockout animals can then be bredto produce more animals.

In another embodiment, oocytes from a sexually mature double knockoutanimal can be in vitro fertilized using wild type sperm from twogenetically diverse pig lines and the embryos implanted into suitablesurrogates. Offspring from these matings can be tested for the presenceof the knockout, for example, they can be tested by cDNA sequencing,PCR, toxin A sensitivity and/or lectin binding. Then, at sexualmaturity, animals from each of these litters can be mated.

In certain methods according to this aspect of the invention,pregnancies can be terminated early so that fetal fibroblasts can beisolated and further characterized phenotypically and/or genotypically.Fibroblasts that lack expression of the alpha-1,3-GT gene can then beused for nuclear transfer according to the methods described herein (seealso Dai et al.) to produce multiple pregnancies and offspring carryingthe desired double knockout.

IV. Types of Genetically Modified Porcine Animals

In one aspect of the present invention, porcine animals are provided inwhich one allele of the alpha-1,3-GT gene is inactivated via a genetictargeting event. In another aspect of the present invention, porcineanimals are provided in which both alleles of the alpha-1,3-GT gene areinactivated via a genetic targeting event. In one embodiment, the genecan be targeted via homologous recombination. In other embodiments, thegene can be disrupted, i.e. a portion of the genetic code can bealtered, thereby affecting transcription and/or translation of thatsegment of the gene. For example, disruption of a gene can occur throughsubstitution, deletion (“knockout”) or insertion (“knockin”) techniques.Additional genes for a desired protein or regulatory sequence thatmodulate transcription of an existing sequence can be inserted.

Pigs that possess two inactive alleles of the alpha-1,3-GT gene are notnaturally occurring. It was surprisingly discovered that whileattempting to knockout the second allele of the alpha-1,3-GT genethrough a genetic targeting event, a point mutation was identified,which rendered the second allele inactive.

Thus, in another aspect of the present invention, the alpha-1,3-GT genecan be rendered inactive through at least one point mutation. In oneembodiment, one allele of the alpha-1,3-GT gene can be rendered inactivethrough at least one point mutation. In another embodiment, both allelesof the alpha-1,3-GT gene can be rendered inactive through at least onepoint mutation. In one embodiment, this point mutation can occur via agenetic targeting event. In another embodiment, this point mutation canbe naturally occurring. In one specific embodiment the point mutationcan be a T-to-G mutation at the second base of exon 9 of thealpha-1,3-GT gene (FIG. 2). Pigs carrying a naturally occurring pointmutation in the alpha-1,3-GT gene allow for the production ofalpha1,3GT-deficient pigs free of antibiotic-resistance genes and thushave the potential to make a safer product for human use. In otherembodiments, at least two, at least three, at least four, at least five,at least ten or at least twenty point mutations can exist to render thealpha-1,3-GT gene inactive. In other embodiments, pigs are provided inwhich both alleles of the alpha-1,3-GT gene contain point mutations thatprevent any expression of functional alpha1,3GT. In a specificembodiment, pigs are provided that contain the T-to-G mutation at thesecond base of exon 9 in both alleles of the alpha-1,3-GT gene (FIG. 2).

Another aspect of the present invention provides a porcine animal, inwhich both alleles of the alpha-1,3-GT gene are inactivated, whereby oneallele is inactivated by a genetic targeting event and the other alleleis inactivated via a naturally occurring point mutation. In oneembodiment, a porcine animal is provided, in which both alleles of thealpha-1,3-GT gene are inactivated, whereby one allele is inactivated bya genetic targeting event and the other allele is inactivated due topresence of a T-to-G point mutation at the second base of exon 9. In aspecific embodiment, a porcine animal is provided, in which both allelesof the alpha-1,3-GT gene are inactivated, whereby one allele isinactivated via a targeting construct directed to Exon 9 (FIG. 6) andthe other allele is inactivated due to presence of a T-to-G pointmutation at the second base of exon 9.

V. Porcine Organs, Tissues, Cells and Cell Lines

The present invention provides, for the first time, viable porcine inwhich both alleles of the alpha 1,3 galactosyltransferase gene have beeninactivated. The invention also provides organs, tissues, and cellsderived from such porcine, which are useful for xenotransplantation.

In one embodiment, the invention provides porcine organs, tissues and/orpurified or substantially pure cells or cell lines obtained from pigsthat lack any expression of functional alpha1,3GT.

In one embodiment, the invention provides organs that are useful forxenotransplantation. Any porcine organ can be used, including, but notlimited to: brain, heart, lungs, glands, brain, eye, stomach, spleen,pancreas, kidneys, liver, intestines, uterus, bladder, skin, hair,nails, ears, nose, mouth, lips, gums, teeth, tongue, salivary glands,tonsils, pharynx, esophagus, large intestine, small intestine, rectum,anus, pylorus, thyroid gland, thymus gland, suprarenal capsule, bones,cartilage, tendons, ligaments, skeletal muscles, smooth muscles, bloodvessels, blood, spinal cord, trachea, ureters, urethra, hypothalamus,pituitary, adrenal glands, ovaries, oviducts, uterus, vagina, mammaryglands, testes, seminal vesicles, penis, lymph, lymph nodes and lymphvessels.

In another embodiment, the invention provides tissues that are usefulfor xenotransplantation. Any porcine tissue can be used, including, butnot limited to: epithelium, connective tissue, blood, bone, cartilage,muscle, nerve, adenoid, adipose, areolar, bone, brown adipose,cancellous, muscle, cartaginous, cavernous, chondroid, chromaffin,dartoic, elastic, epithelial, fatty, fibrohyaline, fibrous, Gamgee,gelatinous, granulation, gut-associated lymphoid, Haller's vascular,hard hemopoietic, indifferent, interstitial, investing, islet,lymphatic, lymphoid, mesenchymal, mesonephric, mucous connective,multilocular adipose, myeloid, nasion soft, nephrogenic, nodal, osseous,osteogenic, osteoid, periapical, reticular, retiform, rubber, skeletalmuscle, smooth muscle, and subcutaneous tissue.

In a further embodiment, the invention provides cells and cell linesfrom porcine animals that lack expression of functional alpha1,3GT. Inone embodiment, these cells or cell lines can be used forxenotransplantation. Cells from any porcine tissue or organ can be used,including, but not limited to: epithelial cells, fibroblast cells,neural cells, keratinocytes, hematopoietic cells, melanocytes,chondrocytes, lymphocytes (B and T), macrophages, monocytes, mononuclearcells, cardiac muscle cells, other muscle cells, granulosa cells,cumulus cells, epidermal cells, endothelial cells, Islets of Langerhanscells, pancreatic insulin secreting cells, pancreatic alpha-2 cells,pancreatic beta cells, pancreatic alpha-1 cells, blood cells, bloodprecursor cells, bone cells, bone precursor cells, neuronal stem cells,primordial stem cells., hepatocytes, keratinocytes, umbilical veinendothelial cells, aortic endothelial cells, microvascular endothelialcells, fibroblasts, liver stellate cells, aortic smooth muscle cells,cardiac myocytes, neurons, Kupffer cells, smooth muscle cells, Schwanncells, and epithelial cells, erythrocytes, platelets, neutrophils,lymphocytes, monocytes, eosinophils, basophils, adipocytes,chondrocytes, pancreatic islet cells, thyroid cells, parathyroid cells,parotid cells, tumor cells, glial cells, astrocytes, red blood cells,white blood cells, macrophages, epithelial cells, somatic cells,pituitary cells, adrenal cells, hair cells, bladder cells, kidney cells,retinal cells, rod cells, cone cells, heart cells, pacemaker cells,spleen cells, antigen presenting cells, memory cells, T cells, B cells,plasma cells, muscle cells, ovarian cells, uterine cells, prostatecells, vaginal epithelial cells, sperm cells, testicular cells, germcells, egg cells, leydig cells, peritubular cells, sertoli cells, luteincells, cervical cells, endometrial cells, mammary cells, follicle cells,mucous cells, ciliated cells, nonkeratinized epithelial cells,keratinized epithelial cells, lung cells, goblet cells, columnarepithelial cells, dopamiergic cells, squamous epithelial cells,osteocytes, osteoblasts, osteoclasts, dopaminergic cells, embryonic stemcells, fibroblasts and fetal fibroblasts. In a specific embodiment,pancreatic cells, including, but not limited to, Islets of Langerhanscells, insulin secreting cells, alpha-2 cells, beta cells, alpha-1 cellsfrom pigs that lack expression of functional alpha-1,3-GT are provided.

Nonviable derivatives include tisssues stripped of viable cells byenzymatic or chemical treatment these tissue derivatives can be furtherprocessed via crosslinking or other chemical treatments prior to use intransplantation. In a preferred embodiment, the derivatives includeextracelluar matrix derived from a variety of tissues, including skin,urinary, bladder or organ submucosal tissues. Also, tendons, joints andbones stripped of viable tissue to include heart valves and othernonviable tissues as medical devices are provided.

Therapeutic Uses

The cells can be administered into a host in order in a wide variety ofways. Preferred modes of administration are parenteral, intraperitoneal,intravenous, intradermal, epidural, intraspinal, intrasternal,intra-articular, intra-synovial, intrathecal, intra-arterial,intracardiac, intramuscular, intranasal, subcutaneous, intraorbital,intracapsular, topical, transdermal patch, via rectal, vaginal orurethral administration including via suppository, percutaneous, nasalspray, surgical implant, internal surgical paint, infusion pump, or viacatheter. In one embodiment, the agent and carrier are administered in aslow release formulation such as a direct tissue injection or bolus,implant, microparticle, microsphere, nanoparticle or nanosphere.

Disorders that can be treated by infusion of the disclosed cellsinclude, but are not limited to, diseases resulting from a failure of adysfunction of normal blood cell production and maturation (i.e.,aplastic anemia and hypoproliferative stem cell disorders); neoplastic,malignant diseases in the hematopoietic organs (e.g., leukemia andlymphomas); broad spectrum malignant solid tumors of non-hematopoieticorigin; autoimmune conditions; and genetic disorders. Such disordersinclude, but are not limited to diseases resulting from a failure ordysfunction of normal blood cell production and maturationhyperproliferative stem cell disorders, including aplastic anemia,pancytopenia, agranulocytosis, thrombocytopenia, red cell aplasia,Blackfan-Diamond syndrome, due to drugs, radiation, or infection,idiopathic; hematopoietic malignancies including acute lymphoblastic(lymphocytic) leukemia, chronic lymphocytic leukemia, acute myelogenousleukemia, chronic myelogenous leukemia, acute malignant myelosclerosis,multiple myeloma, polycythemia vera, agnogenic myelometaplasia,Waldenstrom's macroglobulinemia, Hodgkin's lymphoma, non-Hodgkin'slymphoma; immunosuppression in patients with malignant, solid tumorsincluding malignant melanoma, carcinoma of the stomach, ovariancarcinoma, breast carcinoma, small cell lung carcinoma, retinoblastoma,testicular carcinoma, glioblastoma, rhabdomyosarcoma, neuroblastoma,Ewing's sarcoma, lymphoma; autoimmune diseases including rheumatoidarthritis, diabetes type I, chronic hepatitis, multiple sclerosis,systemic lupus erythematosus; genetic (congenital) disorders includinganemias, familial aplastic, Fanconi's syndrome, dihydrofolate reductasedeficiencies, formamino transferase deficiency, Lesch-Nyhan syndrome,congenital dyserythropoietic syndrome I-IV, Chwachmann-Diamond syndrome,dihydrofolate reductase deficiencies, formamino transferase deficiency,Lesch-Nyhan syndrome, congenital spherocytosis, congenitalelliptocytosis, congenital stomatocytosis, congenital Rh null disease,paroxysmal nocturnal hemoglobinuria, G6PD (glucose-6-phhosphatedehydrogenase) variants 1, 2, 3, pyruvate kinase deficiency, congenitalerythropoietin sensitivity, deficiency, sickle cell disease and trait,thalassemia alpha, beta, gamma, met-hemoglobinemia, congenital disordersof immunity, severe combined immunodeficiency disease (SCID), barelymphocyte syndrome, ionophore-responsive combined immunodeficiency,combined immunodeficiency with a capping abnormality, nucleosidephosphorylase deficiency, granulocyte actin deficiency, infantileagranulocytosis, Gaucher's disease, adenosine deaminase deficiency,Kostmann's syndrome, reticular dysgenesis, congenital Leukocytedysfunction syndromes; and others such as osteoporosis, myelosclerosis,acquired hemolytic anemias, acquired immunodeficiencies, infectiousdisorders causing primary or secondary immunodeficiencies, bacterialinfections (e.g., Brucellosis, Listerosis, tuberculosis, leprosy),parasitic infections (e.g., malaria, Leishmaniasis), fungal infections,disorders involving disproportionsin lymphoid cell sets and impairedimmune functions due to aging, phagocyte disorders, Kostmann'sagranulocytosis, chronic granulomatous disease, Chediak-Higachisyndrome, neutrophil actin deficiency, neutrophil membrane GP-180deficiency, metabolic storage diseases, mucopolysaccharidoses,mucolipidoses, miscellaneous disorders involving immune mechanisms,Wiskott-Aldrich Syndrome, alpha 1-antirypsin deficiency, etc.

Diseases or pathologies include neurodegenerative diseases,hepatodegenerative diseases, nephrodegenerative disease, spinal cordinjury, head trauma or surgery, viral infections that result in tissue,organ, or gland degeneration, and the like. Such neurodegenerativediseases include but are not limited to, AIDS dementia complex;demyeliriating diseases, such as multiple sclerosis and acutetransferase myelitis; extrapyramidal and cerebellar disorders, such aslesions of the ecorticospinal system; disorders of the basal ganglia orcerebellar disorders; hyperkinetic movement disorders, such asHuntington's Chorea and senile chorea; drug-induced movement disorders,such as those induced by drugs that block CNS dopamine receptors;hypokinetic movement disorders, such as Parkinson's disease; progressivesupra-nucleo palsy; structural lesions of the cerebellum;spinocerebellar degenerations, such as spinal ataxia, Friedreich'sataxia, cerebellar cortical degenerations, multiple systemsdegenerations (Mencel, Dejerine Thomas, Shi-Drager, and Machado-Joseph),systermioc disorders, such as Rufsum's disease, abetalipoprotemia,ataxia, telangiectasia; and mitochondrial multi-system disorder;demyelinating core disorders, such as multiple sclerosis, acutetransverse myelitis; and disorders of the motor unit, such as neurogenicmuscular atrophies (anterior horn cell degeneration, such as amyotrophiclateral sclerosis, infantile spinal muscular atrophy and juvenile spinalmuscular atrophy); Alzheimer's disease; Down's Syndrome in middle age;Diffuse Lewy body disease; Senile Demetia of Lewy body type; Parkinson'sDisease, Wernicke-Korsakoff syndrome; chronic alcoholism;Creutzfeldt-Jakob disease; Subacute sclerosing panencephalitishallerrorden-Spatz disease; and Dementia pugilistica. See, e.g., Berkowet. al., (eds.) (1987), The Merck Manual, (15^(th)) ed.), Merck and Co.,Rahway, N.J.

The present invention is described in further detail in the followingexamples. The examples provided below are intended to be illustrativeonly, and are not intended to limit the scope of the invention.

EXAMPLES Example 1 Production of Porcine Cells Heterozygous for theAlpha-1,3-GT Gene

Isolation and transfection of primary porcine fetal fibroblasts. Fetalfibroblast cells (PCFF4-1 to PCFF4-10) were isolated from 10 fetuses ofthe same pregnancy at day 33 of gestation. After removing the head andviscera, fetuses were washed with Hanks' balanced salt solution (HBSS;Gibco-BRL, Rockville, Md.), placed in 20 ml of HBSS, and diced withsmall surgical scissors. The tissue was pelleted and resuspended in50-ml tubes with 40 ml of DMEM and 100 U/ml collagenase (Gibco-BRL) perfetus. Tubes were incubated for 40 min in a shaking water bath at 37° C.The digested tissue was allowed to settle for 3-4 min and the cell-richsupernatant was transferred to a new 50-ml tube and pelleted. The cellswere then resuspended in 40 ml of DMEM containing 10% fetal calf serum(FCS), 1× nonessential amino acids, 1 mM sodium pyruvate and 2 ng/mlbFGF, and seeded into 10 cm. dishes. All cells were cryopreserved uponreaching confluence. SLA-1 to SLA-10 cells were isolated from 10 fetusesat day 28 of pregnancy. Fetuses were mashed through a 60-mesh metalscreen using curved surgical forceps slowly so as not to generateexcessive heat. The cell suspension was then pelleted and resuspended in30 ml of DMEM containing 10% FCS, 1× nonessential amino acids, 2 ng/mlbFGF, and 10 μg/ml gentamycin. Cells were seeded in 10-cm dishes,cultured one to three days, and cryopreserved. For transfections, 10 μgof linearized vector DNA was introduced into 2 million cells byelectroporation. Forty-eight hours after transfection, the transfectedcells were seeded into 48-well plates at a density of 2,000 cells perwell and were selected with 250 μg/ml of G418.

Knockout vector construction Two alpha-1,3-GT knockout vectors, pPL654and pPL657, were constructed from isogenic DNA of two primary porcinefetal fibroblasts, SLA1-10 and PCFF4-2 cells. A 6.8-kb alpha-1,3-GTgenomic fragment, which includes most of intron 8 and exon 9, wasgenerated by PCR from purified DNA of SLA1-10 cells and PCFF4-2 cells,respectively. The unique EcORV site at the 5′ end of exon 9 wasconverted into a SalI site and a 1.8-kb IRES-neo-poly A fragment wasinserted into the SalI site. IRES (internal ribosome entry site)functions as a translation initial site for neo protein. Thus, bothvectors have a 4.9-kb 5′ recombination arm and a 1.9-kb 3′ recombinationarm (FIG. 6).

3′PCR and long-range PCR Approximately 1,000 cells were resuspended in 5μl embryo lysis buffer (ELB) (40 mM Tris, pH 8.9, 0.9% Triton X-100,0.9% NP40, 0.4 mg/ml Proteinase K), incubated at 65° C. for 15 min tolyse the cells and heated to 95° C. for 10 min to inactivate theProteinase K. For 3′ PCR analysis, fragments were amplified using theExpand High Fidelity PCR system (Roche Molecular Biochemicals) in 25 μlreaction volume with the following parameters: 35 cycles of 1 min at 94°C., 1 min at 60° C., and 2 min at 72° C. For LR-PCR, fragments wereamplified by using TAKARA LA system (Panvera/Takara) in 50 μl reactionvolume with the following parameters: 30 cycles of 10 s at 94° C., 30 sat 65° C., 10 min+20 s increase/cycle at 68° C., followed by one finalcycle of 7 min at 68° C. 3′PCR and LR-PCR conditions for purified DNAwas same as cells except that 1 μl of purified DNA (30 μg/ml) was mixedwith 4 μl ELB.

Southern blot analysis of cell samples Approximately 106 cells werelysed overnight at 60° C. in lysis buffer (10 mM Tris, pH 7.5, 10 mMEDTA, 10 mM NaCl, 0.5% (w/v) Sarcosyl, 1 mg/ml proteinase K) and the DNAprecipitated with ethanol. The DNA was then digested with BstEII andseparated on a 1% agarose gel. After electrophoresis, the DNA wastransferred to a nylon membrane and probed with the 3′-enddigoxigenin-labeled probe. Bands were detected using a chemiluminescentsubstrate system (Roche Molecular Biochemicals).

Results Antibiotic (G418) resistant colonies were screened by 3′ PCRwith neo442S and αGTE9A2 as forward and reverse primers. Neo442S is atthe 3′ end of the neo gene and αGTE9A2 is at the 3′ end of exon 9 insequences located outside of the 3′ recombination arm (FIG. 6).Therefore, only through successful targeting at the α1,3GT locus wouldthe expected 2.4 kb PCR product be obtained. From a total of seventransfections in four different cell lines, 1105 G418 resistant colonieswere picked, of which 100 (9%) were positive for α1,3 GT gene disruptionin the initial 3′ PCR screen (range 2.5-12%). Colonies 657A-A8, 657A-I6,and 657A-I11 showed the expected 2.4 kb band, while control PCFF4-6cells, and another G418 resistant colony, 657A-P6, were negative. Aportion of each 3′ PCR positive colony was frozen down immediately, inseveral small aliquots, for future use in NT experiments, while the restof cells were expanded for long-range PCR (LR-PCR) and Southernanalysis.

Since PCR analysis to detect recombination junctions, or mRNA analysis(RT-PCR) can generate false positive results, a long-range PCR, whichwould encompass the entire targeted region, was performed. The LR-PCRcovers the 7.4 kb α1,3GT genomic sequence from exon 8 to the end of exon9, with both primers (aGTE8S and aGTE9A2) located outside of therecombination region (FIG. 2). The control PCFF4-6 cells, and the 3′PCR-negative colony, 657A-P6, showed only the endogenous 7.4 kb bandfrom the wild-type α1,3GT locus. In contrast, three of the 3′ PCRpositive colonies, 657A-A8, 657A-I6 and 657A-I11, showed both the 7.4 kbendogenous band, and a new 9.2 kb band, of the size expected fortargeted insertion of the 1.8 kb IRES-neo cassette into the α1,3GTlocus.

Approximately half (17/30) of the LR-PCR positive colonies weresuccessfully expanded to yield sufficient cell numbers (1×106 cells) forSouthern analysis. It was anticipated that the colonies would beheterozygous for knockout at the α1,3 GT locus, and thus they shouldhave one normal, unmodified gene copy, and one disrupted copy of theα1,3 GT gene. With BstEII digestion, the α1,3 GT knockout cells shouldshow two bands: one 7 kb band of the size expected for the endogenousα1,3 GT allele, and a 9 kb band characteristic of insertion of theIRES-neo sequences at the α1,3 GT locus (FIG. 2). All 17 LR-PCR positivecolonies were confirmed by Southern analysis for the knockout. The samemembranes were re-probed with sequences specific for neo and the 9 kbband was detected with the neo probe, thus confirming targeted insertionof the IRES-neo cassette at the disrupted α1,3GT locus.

Example 2 Production of Porcine Cells Homozygous for the Alpha-1,3-GTGene

Heterozygous alpha-1,3-GT knockout fetal fibroblasts, (657A-I11 1-6)cells, were isolated from a day-32 pregnancy as described above (Seealso Dai et al. Nature Biotechnology 20:451 (2002)). An ATG (startcodon)-targeting alpha-1,3-GT knockout vector was constructed (pPL680),which also contained a neo gene, to knock out the second allele of thealpha-1,3-GT gene. These cells were transfected by electroporation withpPL680 and selected for the alpha1,3Gal-negative phenotype with purifiedC. difficile toxin A (described below).

Example 3 Selection with C. difficile Toxin A for Porcine CellsHomozygous for the Alpha-1,3-GT Gene

Toxin A Cyototoxicity Curve

Porcine cells (PCFF4-6) were exposed for 1 hour or overnight to ten-foldserial dilutions of toxin A (0.00001 μg/ml to 10 μg/ml). Cells werecultured in 24 well plates and were incubated with the toxin for 1 houror overnight at 37° C. The results of this exposure are detailed inTable 2. Clearly, a 1 hour exposure to toxin A at >1 μg/ml resulted in acytotoxic effect on >90% of the cells. A concentration of toxin A at orslightly above 1 μg/ml therefore was chosen for selection of geneticallyaltered cells.

TABLE 2 Toxin A toxicity at 1 hour and overnight exposure [Toxin A],μg/ml 1 hour incubation Overnight incubation  0 100% confluency 100%confluency  .00001 100% confluency 100% confluency  .0001 100%confluency 100% confluency  .001 100% confluency 100% confluency  .01100% confluency  50% confluency, 50% rounded  .1  90% confluency Same as10 ug/ml  1 >90% rounded Same as 10 ug/ml 10 All cells rounded up Allcells rounded up, some lifted

Disaggregated cells from a porcine embryo (I-11: 1-6) which contained apreviously identified targeted knockout in one allele of the galalpha-1,3-GT gene (Dai et al.) were transfected with 10 ug linearizedvector DNA (promoter trap) by electroporation. After 48 hours, the cellswere seeded into 48 well plates at a density of 2000 cells per well andselected with 250 ug/ml G418. Five days post-transfection, media waswithdrawn from the wells, and replaced with 2 ug/ml toxin A in culturemedia (DMEM high glucose with 2.8 ng/ml bFGF and 20% FCS). Cells wereexposed to the selective effect of toxin A for 2 hours at 37 C. Thetoxin A-containing media, along with any affected cells that havereleased from the plate surface, was withdrawn, the remaining cellswashed with fresh media, and the media without toxin A replaced. Tendays later, cells were again exposed to toxin A at 1.3 ug/ml in mediafor 2 hours at 37 C. The media, toxin A, and any cells in solution wereremoved, the remaining cells washed, and the media replaced.

Sixteen days post-transfection, a single colony that exhibited toxin Ainsensitivity, designated 680B1, was harvested and a portion sent forDNA analysis and lectin staining. DNA analysis indicated that the toxinA insensitivity was not due to integration of the second target vector;however, the cells did not stain with GSL IB-4 lectin, indicating that afunctional knockout of the locus had occurred. The 680B1 double knockoutcells were used for nuclear transfer into 5 recipients and threepregnancies resulted. Two of these pregnancies spontaneously aborted inthe first month; the four fetuses from the remaining pregnancy wereharvested on day 39 of the pregnancy and the cells disaggregated andseeded into tissue culture. These fetal cells (680B1-1, 680B1-2,680B1-3, 680B1-4) were exposed to toxin A at 1 ug/ml for 1 hour at 37 C,followed by medium removal, cell washing, and medium replacement withouttoxin A. Fetuses 1, 2, and 4 were not affected by toxin A, whereas mostof the cells from fetus 3 rounded up, indicating that this embryo wassensitive to the cytotoxic effects of the toxin A.

Fetuses 1, 2, and 4 did not bind GS IB4 lectin, as indicated by FACSanalysis (see Table 3), while fetus 3 did bind lectin. This suggeststhat fetuses 1, 2, and 4 do not carry the epitope alpha 1,3 gal forwhich this particular lectin is specific.

TABLE 3 FACS Results of 680B1-1 to 680B1-4 Cells with GS-IB4 Lectin GSIB4 lectin positive cells (%) 50 μg/ml IB4 100 μg/ml Cell Unstaininglectin IB4 lectin HeLa Cells (Negative   1%   2% 2.8% CTL) PCFF4-6 cells(Positive 0.2%  76%  91% CTL) PFF4 cells (Positive 1.5%  82%  94% CTL)680B1-1 cells 0.6% 0.8% 0.9% 680B1-2 cells 1.2% 1.2% 1.1% 680B1-3 cells  8%  35%  62% 680B1-4 cells 0.6% 0.8% 0.9%

A complement fixation assay was run on cells from all four fetuses. Thecomplement lysis assay was developed as a bioassay for lack of alpha galexpression. Human serum contains high levels of pre-formed antibodyagainst alpha gal as well as the full portfolio of complement regulatoryproteins (the C3 pathway). The presence of alpha gal on the surface of acell, upon binding of anti-alpha gal antibody, activates the complementcascade, and results in complement-mediated cell lysis. Alpha-galnegative cells would be resistant to complement mediated lysis. In threeseparate tests, B1 and control pig cells were exposed to human serumplus complement, and assays performed to evaluate sensitivity orresistance to alpha-gal-initiated, complement-mediated cell lysis. Theassay was performed with B1-1, B1-2, and B1-4 cells, as well asheterozygous GT KO cells (B1-3, gal positive), and with wild-typealpha-gal (+) PCFF4-6 pig cells as a control. Cells were exposed to oneof three treatments; two negative controls, bovine serum albumin (BSA),and heat-inactivated human serum (HIA-HS) do not contain any functionalcomplement protein and thus would not be expected to cause anysignificant cell lysis; the third treatment, non-heat-inactivated humanserum (NHS) contains functional human complement as well as anti-galspecific antibodies, and thus would be expected to lyse cells which havegalactose alpha 1,3 galactose on their cell surface.

The results shown in FIG. 1 clearly demonstrate that B1-1, B-2 and B 1-4cells are resistant to human complement-mediated lysis while B 1-3cells, which is a 1,3 Gal positive, is still as sensitive to humanplasma as are wild-type PCFF4-6 cells.

Sequencing results of cDNA from all fetuses indicated that fetuses 1,2and 4 contain a point mutation in the second alpha 1,3 GT allele, achange that could yield a dysfunctional enzyme (see FIG. 2). Thismutation occurred at bp424 of the coding region, specifically, thesecond base pair of exon 9, of the alpha-1,3-GT (GGTA1) gene (GenBankAccession No.L36152) as a conversion of a thymine to a guanine residue,which results in an amino acid substitution of tyrosine at aa 142 to anaspartic acid.

This is a significant conversion, as the tyrosine, a hydrophilic aminoacid, is a critical component of the UDP binding site of alpha 1,3GT(see FIG. 3). Analysis of the crystal structure of bovine alpha-1,3-GTprotein showed that this tyrosine is the center of the catalytic domainof the enzyme, and is involved in UDP-Gal binding (Gastinel et. al.,EMBO Journal 20(4): 638-649, 2001). Therefore, a change from tyrosine (ahydrophobic amino acid) to aspartic acid (a hydrophilic amino acid)would be expected to cause disruption of the αGT function (as observed).

To confirm that the mutated cDNA will not make functional αGT protein,the cDNAs from the second allele of all 4 cells were cloned into anexpression vector and this GT expression vector transfected into humanfibroblast cells (HeLa cells) as well as into primary Rhesus monkeycells. As humans and Old World monkeys lack a functional alpha 1,3 GTgene, the HeLa cells would not have an alpha 1,3 galactose on their cellsurface (as assayed by lectin binding experiments). Results showed thatthe HeLa and monkey cells, when transfected with cDNA obtained fromB1-1, B1-2 and B1-4 cells, were still α1,3 Gal negative by IB4-lectinstaining, while Hela and Rhesus monkey cells transfected with cDNA fromthe B1-3, made a functional alpha 1,3 GT transcript and subsequentlywere α1,3Gal positive. Clearly, cells with the aspartate mutation(instead of tyrosine) cannot make functional alpha 1,3 galactosyltransferase

Example 4 Generation of Cloned Pigs Using Homozygous Alpha 1,3GT-Deficient Fetal Fibroblasts as Nuclear Donors

Preparation of cells for nuclear transfer. Donor cells were geneticallymanipulated to produce cells homozygous for alpha 1,3 GT deficiency asdescribed generally above. Nuclear transfer was performed by methodsthat are well known in the art (see, e.g., Dai et al., NatureBiotechnology 20: 251-255, 2002; and Polejaeva et al., Nature 407:86-90,2000), using toxin A-selected porcine fibroblasts as nuclear donors thatwere produced as described in detail hereinabove

Embryo transfers and resulting live births. In the initial attempt toproduce live alpha-1,3-GT dKO pigs by nuclear transfer, a total of 16embryo transfers were performed with genetically manipulated donorcells. Nine initial pregnancies were established but only two wentbeyond Day 75 of gestation. Five piglets were born on the Jul. 25, 2002.One piglet died immediately after birth and another four were born aliveand appeared normal (FIG. 4).

Example 5 Analysis of Homozygous Alpha 1,3 GT Knockout Pigs

Tail fibroblast cells and umbilicus tissue sections were obtained fromall 5 double knockout piglets and stained using the GS-IB4 lectin asdescribed previously. No staining was observed, indicating a completelack of galactose alpha 1,3 galactose epitope on the surface of tissuesfrom these animals (data not shown). Aorta endothelial cells and muscleand tail fibroblasts isolated from the dead piglet (761-1) were negativewith GS-IB4 lectin staining. FACS analysis of muscle fibroblasts frompiglet 761-1 also showed a negative result for GS-IB4 binding. Tissuesections of liver, kidney, spleen, skin, intestine, muscle, brain,heart, pancreas, lung, aorta, tongue, umbilicus, and tail obtained frompiglet 761-1 were all negative with GS-IB4 staining, indicating acomplete lack of detectable cell surface alpha 1,3Gal epitopes (Phelpset al., Science 299: 411-414, 2003 including figure S3).

We performed an in vivo immunogenicity test with alpha 1,3GT-knockoutmice. We injected islet-like cell clusters (ICCs) isolated from thepancreas of piglet 761-1 intraperitoneally into alpha 1,3GT knockoutmice. We used ICCs from a neonatal wild-type piglet as a control. Asshown in FIG. 5, no increase in the titer of immunoglobulin M (IgM) toalpha 1,3Gal was observed in alpha 1,3GT knockout mice after injectionwith ICCs from the alpha 1,3GT DKO piglet, in contrast to significantIgM titer increases observed in those mice injected with wild-typepiglet ICCs (Phelps et al., Science 299: 411-414, 2003 including figureS4). This result clearly demonstrates that the DKO piglet cells do notmake any alpha 1,3Gal epitopes.

Sequencing of DNA obtained from all five piglets confirmed the presenceof the mutation at bp 424 of the GGTA1 gene, as observed in the 680B1-2cells used to clone these animals (FIG. 2).

Since this first successful production of a litter of alpha-GT dKO pigs,two subsequent litters of dKO piglets have been produced by nucleartransfer, in one case (litter 662) using the dKO fetal fibroblasts asnuclear donor cells. Litter 660 was produced by nuclear transfer usingtail fibroblast cells from a member of the litter 761 as nuclear donor.These births are summarized in Table 4.

TABLE 4 Summary of alpha-GT double knockout births produced by nucleartransfer Litter ID Nuclear Donor No. Births Live Births 761 680B:1-2 5 4662 680B:1-2 1 0 660 761-5 4 2

Example 6 Breeding of Heterozygous Alpha 1,3 GT Single Knockout (SKO)Male and Female Pigs to Establish a Miniherd of Double Knockout (DKO)Pigs

A total of 29 Southern blot confirmed cloned GT-SKO females and 25Southern blot confirmed GT-SKO male cloned pigs have been generated todate. These male and female heterozygous (single gene alpha1,3GTknockout pigs) have been bred by natural breeding and by artificialinsemination(AI), in order to generate a herd of DKO pigs for use inpreclinical studies and human clinical trials. We have produced 16alpha1,3-GT DKO piglets from 13 litters.

This invention has been described with reference to illustrativeembodiments. Other embodiments of the general invention described hereinand modifications there of will be apparent to those of skill in the artand are all considered within the scope of the invention.

1. A method for the production of a pig that lacks any expression offunctional alpha-1,3 galactosyltransferase (α-1,3 GT) comprising:breeding a male pig heterozygous for an inactivation of the alpha-1,3 GTgene with a female pig heterozygous for an inactivation of the α-1,3 GTgene, wherein one or both pigs are heterozygous due to the presence of apoint mutation in an allele of the α-1,3 GT gene, wherein the pointmutation is a T-to-G point mutation at the second base of exon 9 of theα-1,3 GT gene or a point mutation induced or produced by a geneticmodification in the α-1,3 GT gene.
 2. The method of claim 1, wherein thepoint mutation is a T-to-G point mutation at the second base of exon 9of the α-1,3 GT gene.
 3. The method of claim 1, wherein a male pig thatcontains a T-to-G point mutation at the second base of exon 9 of theα-1,3 GT gene is bred with a female pig that contains a T-to-G pointmutation at the second base of exon 9 of the α-1,3 GT gene.
 4. Themethod of claim 1, wherein at least one of the male or female pigscomprises at least one allele of an α-1,3 GT gene that is inactivatedvia a genetic targeting event.
 5. The method of claim 1, furthercomprising breeding together the pigs that lack any expression offunctional α-1,3 GT to produce a herd of pigs that lack any expressionof functional α-1,3 GT.
 6. A non-naturally occurring pig producedaccording to the method of claim 1, 2, 3, 4 or
 5. 7. A cell, tissue, ororgan obtained from the pig of claim
 6. 8. The cell, tissue or organ ofclaim 7 wherein at least one allele of an α-1,3 GT gene is inactivatedvia a genetic targeting event.